ONT Long-Read Transcriptomics (IsoQuant)
Barcode 24 analysis
Type
CWL
Status
succeeded
Engine
cwltool
Duration
0.5 h
Source Data
| Study | Strain-specific cortex gene expression and isoform usage |
| Sample prep | PCR-cDNA barcoding library preparation for C57/DBA mouse RNA (run 3, bc10-15) 2023-05-09 |
| Sequencing | Nanopore PCR-cDNA sequencing of C57/DBA mouse RNA (run 3) 2023-05-12 |
| Run Data | Run #63 (6 samples) |
| Samples | C57_rep1_bc10 C57_rep2_bc11 C57_rep3_bc12 DBA_rep1_bc13 DBA_rep2_bc14 DBA_rep3_bc15 |
Pipeline
ONT Long-Read Transcriptomics (IsoQuant)
Run #46
(this run)
succeeded
ONT Isoform Functional Annotation (superseded)
Sample Provenance
Process Chain
1
Dissection and collection of tissue from 10-week-old male mice (C57/BL6J and DBA/2J)
(2016-10-17)
Labbook
Protocol: Dissection and collection of tissue from 10-week-old male mice
8 samples produced
2
Dissection and collection of tissue from 10-week-old male mice (C57/BL6J and DBA/2J) continued
(2016-10-17)
Labbook
Protocol: Dissection and collection of tissue from 10-week-old male mice
24 samples produced
3
Simultaneous extraction of DNA and RNA from C57 and DBA cortex tissue using Qiagen AllPrep
(2016-11-17)
Labbook
Protocol: Simultaneous DNA and RNA extraction from animal tissues using QIAGEN AllPrep DNA/RNA Mini Kit
16 samples produced
4
PCR-cDNA barcoding library preparation for C57/DBA mouse RNA (SQK-PCB111.24)
(2023-03-13)
Labbook
Protocol: PCR-cDNA Barcoding sequencing library preparation (SQK-PCB111.24)
6 samples produced
5
Fragment Analyzer analysis of C57/DBA PCR-cDNA libraries (pre-selection)
(2023-03-14)
Labbook
24 measurements across 6 samples
6
PCR-cDNA barcoding library preparation for C57/DBA mouse RNA (run 3, bc10-15)
(2023-05-09)
Labbook
Protocol: PCR-cDNA Barcoding sequencing library preparation (SQK-PCB111.24)
6 samples produced
7
Nanopore PCR-cDNA sequencing of C57/DBA mouse RNA (run 3)
(2023-05-12)
Labbook
QC Measurements
Fragment Analyzer analysis of C57/DBA PCR-cDNA libraries (pre-selection)
2023-03-14
Labbook
| Sample | bp | count | ng/µL | nmol/L |
|---|---|---|---|---|
| C57_rep1_bc19 | 1493 | 3 | 5.6008 | 6.1563 |
| C57_rep2_bc20 | 1493 | 4 | 6.2061 | 7.4416 |
| C57_rep3_bc21 | 1485 | 3 | 5.7241 | 6.8672 |
| DBA_rep1_bc22 | 1500 | 2 | 4.2355 | 4.4695 |
| DBA_rep2_bc23 | 1485 | 2 | 4.0452 | 4.2648 |
| DBA_rep3_bc24 | 1485 | 2 | 4.3327 | 4.7107 |
Workflow
ONT Transcriptomics (IsoQuant + TransDecoder)
#cwl
Software Tools
| Tool | Version | URL |
|---|---|---|
| cwltool | - | https://github.com/common-workflow-language/cwltool |
| isoquant_3.6.0--hdfd78af_0.sif | - | - |
Results Summary
Total Reads
935,756
Expressed Genes (CPM ≥ 1)
12,296
Novel Transcripts (CPM ≥ 1)
1,003
PolyA Detected
88.4%
rRNA Reads
0.1%
mRNA Genes (CPM ≥ 1)
12,295
mRNA Transcripts (CPM ≥ 1)
17,900
ORFs (mRNA only)
12,225
Expressed Transcript Sources (CPM ≥ 1, unique reads, rRNA excluded)
Novel Transcript Categories (CPM ≥ 1, unique reads, rRNA excluded)
Top Expressed Genes (CPM, unique reads)
All Reads (inc. rRNA)
mRNA Only (rRNA excluded)
Gene Expression Distribution (CPM, unique reads)
All Reads (inc. rRNA)
mRNA Only (rRNA excluded)
Read Assignment Quality
Read Structural Classification
Chromosomal Distribution (unique reads)
All Unique Reads
mRNA Reads Only
Gene Biotype Distribution (expressed genes, CPM ≥ 1, rRNA excluded)
Transcript Length Distribution (expressed transcripts, CPM ≥ 1, rRNA excluded)
11,995 transcripts | Median: 1,802 nt | Mean: 2,202 nt | N50: 2,706 nt
Exons per Transcript (expressed transcripts, CPM ≥ 1, rRNA excluded)
Isoforms per Gene (expressed transcripts, CPM ≥ 1, rRNA excluded)
TransDecoder ORF Types (expressed transcripts, rRNA excluded)
Peptide Length Distribution (expressed transcripts, rRNA excluded)
12,225 ORFs | Median: 273 aa | Mean: 356 aa | Max: 4,967 aa
Output Files
Input Data
| File | Format | Description |
|---|---|---|
OUT.transcript_models.gtf |
text/plain | - |
OUT.extended_annotation.gtf |
text/plain | - |
OUT.read_assignments.tsv.gz |
application/octet-stream | - |
OUT.transcript_counts.tsv |
text/tab-separated-values | - |
OUT.gene_counts.tsv |
text/tab-separated-values | - |
Provenance
| Execution | Expression quantification summary |
| Completed | 2026-03-01T02:34:58+00:00 |
RO-Crate 1.1
Workflow RO-Crate 1.0
FAIR
This analysis is packaged as a Research Object Crate
with machine-readable provenance and FAIR metadata.
RO-Crate Metadata (JSON-LD)
Show/hide raw JSON-LD
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"text": "# SOP: Dissection and Collection of Tissue from 10-Week-Old Male Mice\n\n**Applies to:** 10-week-old male mice (C57BL/6J, DBA/2J, or other inbred strains)\n**Tissues collected:** Cortex, pituitary, liver\n**Performed by:** Surgical team, The Jackson Laboratory, Bar Harbor, ME, USA\n**Shipping:** Dry shipper via FedEx International Priority to Australia\n\n---\n\n## Document Control\n\n* **SOP ID:** __________\n* **Version:** 1.0\n* **Effective Date:** __________\n* **Review Due:** __________\n* **Author:** __________\n* **Approved By:** __________\n\n**Change History**\n\n| Version | Date | Description | Author |\n| ------- | ---------- | --------------- | ---------- |\n| 1.0 | __________ | Initial release | __________ |\n\n---\n\n## Purpose\n\nTo obtain high-quality, snap-frozen tissue samples (cortex, pituitary, liver) from 10-week-old male mice for downstream molecular analysis (RNA extraction, DNA extraction, proteomics). Tissues are collected at The Jackson Laboratory and shipped internationally to Australia under cryogenic conditions to preserve nucleic acid and protein integrity.\n\n## Scope\n\nAll personnel involved in the planning, coordination, dissection, tissue collection, and shipment of mouse tissue samples between The Jackson Laboratory (USA) and the receiving laboratory (Australia).\n\n## Responsibilities\n\n* **Principal Investigator (receiving lab):** Define experimental design, strain requirements, tissue list, and sample labelling scheme. Coordinate import permits and institutional approvals.\n* **Jackson Laboratory surgical team:** Perform euthanasia, dissection, tissue collection, snap-freezing, and packaging for shipment.\n* **Shipping coordinator:** Arrange FedEx International Priority shipment, customs documentation, and dry shipper logistics.\n* **Receiving lab personnel:** Receive shipment, verify sample integrity, transfer to long-term storage, and log receipt.\n\n## Regulatory and Ethical Requirements\n\n* All animal procedures must be approved by The Jackson Laboratory IACUC (Institutional Animal Care and Use Committee) under an active protocol.\n* Receiving institution must hold a valid **import permit** for biological materials from the relevant national authority (e.g., Australian Department of Agriculture, Fisheries and Forestry \u2014 DAFF).\n* Ensure compliance with CITES regulations (not typically applicable for laboratory mice, but verify for specific strains).\n* **Biosecurity:** Frozen tissues from SPF (Specific Pathogen Free) mice. Health monitoring reports from Jackson Laboratory should accompany the shipment.\n\n---\n\n## Materials and Equipment\n\n### At Jackson Laboratory (dissection)\n\n* Certified CO\u2082 euthanasia station or injectable anaesthetic (as per IACUC protocol)\n* Cervical dislocation tools (secondary method of euthanasia)\n* Sterile surgical instruments: fine scissors, forceps (curved and straight), scalpel with #10 and #15 blades, micro-dissecting forceps, bone rongeurs (for skull)\n* Sterile surgical drapes and pads\n* Sterile PBS (phosphate-buffered saline), ice-cold\n* Liquid nitrogen in Dewar flask\n* Pre-labelled cryovials (1.5\u20132.0 mL, cryogenic-rated, screw-cap)\n* Cryogenic labels (resistant to LN\u2082 and \u221280 \u00b0C)\n* RNAlater (optional, as backup for tissues not immediately snap-frozen)\n* Absorbent pads and biohazard waste bags\n* PPE: lab coat, gloves, face shield (for LN\u2082 handling)\n\n### For shipping\n\n* **Dry shipper** (vapour-phase liquid nitrogen dewar, e.g., MVE SC 4/2V or Taylor-Wharton CX100)\n* Cryogenic storage canes or racks\n* Secondary containment bags (leak-proof, absorbent material)\n* FedEx International Priority shipping account\n* Customs documentation: commercial invoice, packing list, import permit, health certificate, AQIS declaration\n* Temperature data logger (optional but recommended)\n\n### At receiving laboratory (Australia)\n\n* \u221280 \u00b0C ultra-low freezer with allocated space\n* Cryogenic gloves and face shield\n* Sample inventory system / database\n\n---\n\n## Procedure\n\n### 1. Pre-Dissection Planning\n\n1. Confirm **mouse strain, sex (male), and age (10 weeks \u00b1 2 days)** with Jackson Laboratory.\n2. Provide Jackson Laboratory with:\n * Number of animals required (biological replicates)\n * Tissue list: **cortex, pituitary, liver**\n * Sample labelling scheme (e.g., strain_tissue_replicate: C57_CTX_1, DBA_PIT_3)\n * Any special handling instructions (e.g., morning collection to control circadian effects)\n3. Confirm **IACUC protocol** is active and covers the planned procedures.\n4. Obtain **Australian import permit** (DAFF) for frozen animal tissues. Allow 4\u20136 weeks lead time.\n5. Arrange **FedEx International Priority** account and confirm dry shipper availability and return logistics.\n\n### 2. Animal Preparation\n\n1. House mice under standard conditions (12 h light/dark cycle, ad libitum food and water, 20\u201324 \u00b0C) for at least 1 week prior to collection to minimise stress-related transcriptomic changes.\n2. Record for each animal:\n * Strain and stock number\n * Date of birth and age at collection\n * Body weight\n * Cage number and housing conditions\n * Any health observations or abnormalities\n3. Fast animals for **4\u20136 hours** prior to collection if liver metabolomic consistency is required (optional \u2014 confirm with PI).\n\n### 3. Euthanasia\n\n1. Euthanise by **CO\u2082 inhalation** (gradual displacement, 30\u201370% chamber volume/min) followed by **cervical dislocation** as secondary confirmation, in accordance with AVMA Guidelines and IACUC protocol.\n2. Confirm death: absence of heartbeat, respiration, and pedal reflex.\n3. Record **time of death** \u2014 proceed to dissection immediately (within 5 min) to minimise RNA degradation.\n\n### 4. Tissue Dissection and Collection\n\nPerform all dissections on a clean, sterile surface. Use separate sterile instruments for each tissue to prevent cross-contamination. Work quickly \u2014 total dissection time per animal should be **<15 minutes**.\n\n#### 4a. Brain \u2014 Cortex\n\n1. Decapitate the mouse. Remove the skin overlying the skull.\n2. Using bone rongeurs or fine scissors, carefully open the skull along the sagittal suture from the foramen magnum to the olfactory bulbs.\n3. Gently lift and peel back the skull plates to expose the brain.\n4. Using curved forceps, carefully lift the brain from the cranial cavity, severing cranial nerves and the spinal cord at the brainstem.\n5. Place the brain dorsal-side up on a cold surface (glass plate on ice or cold PBS-dampened filter paper).\n6. Using a scalpel or razor blade, remove the **cerebral cortex** by making a coronal cut posterior to the olfactory bulbs and peeling the cortical hemispheres away from the underlying structures (hippocampus, striatum, thalamus).\n7. Transfer cortex tissue immediately to a **pre-labelled cryovial**.\n8. **Snap-freeze** by plunging the cryovial into liquid nitrogen. Hold submerged for \u226530 seconds.\n\n#### 4b. Pituitary\n\n1. After removing the brain, examine the base of the skull (sella turcica).\n2. The **pituitary gland** is a small (~1 mm), pinkish structure sitting in the sella turcica.\n3. Using fine-tipped forceps, gently tease the pituitary free from the surrounding membrane and bone.\n4. Transfer to a **pre-labelled cryovial** and snap-freeze in liquid nitrogen immediately.\n5. **Note:** The pituitary is very small \u2014 take care not to lose or fragment it. If it fragments, collect all pieces.\n\n#### 4c. Liver\n\n1. Open the abdominal cavity with a midline ventral incision from pelvis to sternum.\n2. Retract the intestines to expose the liver.\n3. Identify the **left lateral lobe** (largest, most accessible lobe).\n4. Excise a **~50\u2013100 mg piece** (approximately 5 \u00d7 5 \u00d7 3 mm) from the left lateral lobe using a clean scalpel or razor blade.\n5. Briefly blot on sterile filter paper to remove excess blood.\n6. Transfer to a **pre-labelled cryovial** and snap-freeze in liquid nitrogen.\n7. If the entire liver is required, excise all lobes, record total liver weight, and snap-freeze in separate vials.\n\n### 5. Post-Dissection\n\n1. Transfer all snap-frozen cryovials from liquid nitrogen to a **\u221280 \u00b0C freezer** within 1 hour of collection.\n2. Dispose of carcasses and biological waste according to Jackson Laboratory institutional procedures.\n3. Clean and decontaminate all instruments and work surfaces.\n4. Complete the **dissection log** for each animal:\n\n | Field | Record |\n |---|---|\n | Animal ID | Strain, stock #, ear tag/toe clip |\n | Date and time of euthanasia | |\n | Body weight (g) | |\n | Tissues collected | Cortex, pituitary, liver |\n | Tissue appearance | Normal / abnormal (describe) |\n | Cryovial labels | List all vial IDs |\n | Operator | Name and signature |\n | Notes | Any deviations or observations |\n\n### 6. Packaging for International Shipment\n\n1. **Charge the dry shipper** with liquid nitrogen at least 24 hours before packing. Drain excess liquid \u2014 the dry shipper must contain only **vapour-phase nitrogen** (no free liquid) for air transport compliance (IATA).\n2. Verify the dry shipper holds temperature below **\u2212150 \u00b0C** and has a rated hold time sufficient for transit (typically 10\u201314 days).\n3. Place cryovials in **cryogenic canes or racks** and insert into the dry shipper.\n4. Seal the dry shipper and place in the **outer shipping container** (insulated box or hard case).\n5. Include **absorbent material** inside the secondary containment bag (regulatory requirement for biological specimens).\n6. Attach a **temperature data logger** (optional but recommended) to monitor transit conditions.\n7. Prepare shipping documentation:\n * **Commercial invoice** (contents: frozen mouse tissue samples, no commercial value / research use only)\n * **Packing list** (number of vials, tissue types, strain, animal IDs)\n * **Australian import permit** (DAFF-issued)\n * **Health monitoring report** from Jackson Laboratory (SPF status)\n * **Dangerous goods declaration** \u2014 Class 9 UN3373 Biological Substance Category B (if required by carrier) or exempt if shipped as non-infectious, non-hazardous research specimens in a dry shipper\n * **IATA shipper's declaration** for magnetized material (dry shipper contains no free liquid but is classified as a cryogenic vessel)\n\n### 7. Shipment\n\n1. Schedule **FedEx International Priority** pickup from Jackson Laboratory (Bar Harbor, ME, USA).\n2. Ship **Monday\u2013Wednesday** to avoid weekend delays at customs.\n3. Estimated transit time: **3\u20135 business days** (Bar Harbor \u2192 Australian destination).\n4. Provide the receiving lab with the **tracking number** and expected arrival date.\n5. Alert the receiving lab to arrange customs clearance and immediate collection upon arrival.\n\n### 8. Receipt at Australian Laboratory\n\n1. **Collect the shipment immediately** upon arrival \u2014 do not allow it to sit at the depot.\n2. Open the dry shipper and verify:\n * All cryovials are present and match the packing list\n * Vials are still frozen (frost visible on vials)\n * No signs of thawing (condensation, liquid in vials)\n * Temperature data logger reading (if included) \u2014 confirm temperature remained below \u2212150 \u00b0C throughout transit\n3. Transfer all cryovials immediately to a **\u221280 \u00b0C freezer**.\n4. Log receipt in the laboratory sample inventory:\n\n | Field | Record |\n |---|---|\n | Date received | |\n | Shipment tracking # | |\n | Dry shipper condition | Frost present / temperature OK |\n | Number of vials received | |\n | Vials match packing list? | Yes / No (note discrepancies) |\n | Storage location | Freezer ID, shelf, box, position |\n | Received by | Name and signature |\n\n5. Return the **dry shipper** to Jackson Laboratory per agreed logistics (typically FedEx return shipment).\n\n---\n\n## Quality Control\n\n* **RNA integrity:** Downstream RNA extraction should target RIN/RQN \u2265 7.0 for all tissues. Pituitary may yield lower due to small size \u2014 RQN \u2265 5.0 acceptable.\n* **Dissection time:** Total time from euthanasia to snap-freeze should be **<15 minutes per animal**. Record actual times.\n* **Freeze-thaw:** Tissues must not undergo any freeze-thaw cycles before processing. Monitor freezer temperature logs.\n\n## Troubleshooting\n\n| Problem | Action |\n|---|---|\n| Pituitary fragmented or lost | Record in log; may need to pool from multiple animals or exclude |\n| Tissue partially thawed on arrival | Record in log; process immediately for extraction; note in downstream QC |\n| Customs delay >48 h | Contact FedEx and customs broker; verify dry shipper hold time is sufficient |\n| Dry shipper running low | If hold time is marginal, arrange emergency transfer to local \u221280 \u00b0C |\n| Import permit expired | Do not ship; renew permit before proceeding (allow 4\u20136 weeks) |\n| Animal health abnormality | Record observation; PI to decide whether to include or exclude the animal |\n\n## Waste Disposal\n\n* Mouse carcasses: dispose per Jackson Laboratory IACUC-approved procedures (incineration or licensed biological waste contractor).\n* Sharps (scalpel blades, needles): sharps container.\n* Liquid nitrogen: allow to evaporate in well-ventilated area. Do not pour down drains.\n\n## Records to Maintain\n\n* Approved IACUC protocol number\n* Australian import permit (DAFF) \u2014 copy\n* Dissection logs for each animal\n* Shipping documentation (commercial invoice, packing list, tracking number)\n* Temperature logger data (if used)\n* Sample receipt log\n* Freezer storage map / inventory database entry\n\n---\n\n**End of SOP**"
}
]
},
{
"@id": "#protocol-40",
"@type": "LabProtocol",
"name": "Simultaneous DNA and RNA extraction from animal tissues using QIAGEN AllPrep DNA/RNA Mini Kit",
"description": "Simultaneous purification of genomic DNA and total RNA from the same tissue sample (\u226430 mg) using selective AllPrep DNA spin columns and RNeasy silica membrane technology. Includes on-column DNase treatment for RNA.",
"url": {
"@id": "https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/multianalyte-and-virus/allprep-dna-rna-mini-kit"
},
"version": "1.0",
"category": "sample_prep",
"step": [
{
"@type": "HowToStep",
"position": 1,
"text": "# SOP: Simultaneous DNA and RNA Extraction from Animal Tissues using QIAGEN AllPrep DNA/RNA Mini Kit\n\n**Applies to:** Fresh or frozen animal tissues \u226430 mg per spin column\n**Kit:** QIAGEN AllPrep DNA/RNA Mini Kit (Cat. No. 80204)\n**Location:** __________\n\n---\n\n## Document Control\n\n* **SOP ID:** __________\n* **Version:** 1.0\n* **Effective Date:** __________\n* **Review Due:** __________\n* **Author:** __________\n* **Approved By:** __________\n\n**Change History**\n\n| Version | Date | Description | Author |\n| ------- | ---------- | --------------- | ---------- |\n| 1.0 | __________ | Initial release | __________ |\n\n---\n\n## Purpose\n\nSimultaneous purification of genomic DNA and total RNA from the same tissue sample using the AllPrep DNA spin column (selective DNA binding in high-salt buffer) and the RNeasy spin column (silica membrane RNA binding). This enables paired DNA/RNA analysis from a single biopsy or tissue aliquot.\n\n## Scope\n\nAll personnel performing dual DNA/RNA extraction from animal tissues in this laboratory.\n\n## Responsibilities\n\n* **Operators:** Follow this SOP, record batch details, and maintain RNase-free practice.\n* **Supervisors:** Ensure training, PPE compliance, and kit maintenance.\n\n## Safety and RNase-Free Practice\n\n* Wear lab coat, gloves, and eye protection.\n* Work in a clean area. Treat benches and tools with RNase decontamination solution.\n* \u03b2-mercaptoethanol is toxic and has a strong odor. Handle in a fume hood.\n* Use RNase-free tubes, tips, and reagents only. Change gloves frequently.\n* Buffer RLT Plus contains guanidine isothiocyanate \u2014 harmful if swallowed or in contact with skin. Do not mix with bleach.\n\n## References\n\n* QIAGEN AllPrep DNA/RNA Mini Handbook, November 2020.\n* NASA GeneLab SOP GL-SOP-3.1 v1.0.\n\n---\n\n## Materials and Equipment\n\n**Provided in kit (Cat. No. 80204, 50 preps)**\n\n* Buffer RLT Plus\n* Buffer RW1\n* Buffer RPE (concentrate \u2014 add ethanol before first use)\n* Buffer AW1\n* Buffer AW2\n* AllPrep DNA Mini Spin Columns\n* RNeasy Mini Spin Columns\n* Collection Tubes (1.5 mL and 2 mL)\n* RNase-free Water\n\n**Not supplied**\n\n* \u03b2-mercaptoethanol (\u03b2-ME)\n* 96\u2013100% ethanol\n* 70% ethanol (freshly prepared)\n* RNase-free DNase Set (Cat. No. 79254) \u2014 for on-column DNase treatment\n* RNase/DNase-free 1.5 mL tubes\n* Microcentrifuge capable of \u226512,000 \u00d7 g\n* Homogenization tools: rotor-stator homogenizer, or TissueLyser with stainless steel beads (3\u20137 mm), or RNase-free pestle, or mortar and pestle with liquid nitrogen\n* Optional: RNase-free syringe with 20-gauge needle\n\n---\n\n## Preparations (before each extraction session)\n\n1. **Prepare Buffer RPE** (first use only)\n * Add **4 volumes of 96\u2013100% ethanol** to the concentrate. Mark the bottle to indicate ethanol has been added.\n\n2. **Prepare fresh Lysis Buffer**\n * Add **10 \u00b5L \u03b2-mercaptoethanol per 1 mL Buffer RLT Plus**. Mix well. Prepare only what you need; discard unused buffer.\n\n3. **Prepare DNase Mix** (per sample)\n * Dissolve lyophilized DNase I (1500 Kunitz units) in **550 \u00b5L** RNase-free water. Mix gently. Aliquot and store at \u221220 \u00b0C (stable up to 9 months; thawed aliquots stable at 2\u20138 \u00b0C for 6 weeks).\n * Per sample: **10 \u00b5L DNase I stock + 70 \u00b5L Buffer RDD**. Mix by gentle inversion \u2014 **do not vortex**.\n\n4. **Pre-chill microcentrifuge** to 4 \u00b0C if required for lysate clarification.\n\n5. **Heat 2 mL RNase/DNase-free water** to 70 \u00b0C for DNA elution (optional, improves yield).\n\n6. **Input tissue mass**\n\n | Tissue type | Maximum input |\n |---|---|\n | Standard soft tissue | \u226430 mg |\n | Muscle, heart, skin | \u226415 mg (LN\u2082-preserved) or \u226410 mg (RNAlater) |\n | Liver | \u226420 mg |\n\n---\n\n## Procedure\n\n### 1. Tissue Disruption and Homogenization\n\n1. Weigh tissue (\u226430 mg). Place in a pre-chilled RNase-free tube on ice.\n2. Add **600 \u00b5L Buffer RLT Plus** (with \u03b2-ME).\n3. Homogenize immediately using one of the following:\n\n **Option A \u2014 Rotor-stator:**\n Homogenize for **30\u201345 s** until no visible tissue pieces remain.\n\n **Option B \u2014 TissueLyser / bead mill:**\n Add a single **5 mm stainless steel bead**. Run at **25 Hz for 2 \u00d7 2 min** (rotate adapter halfway).\n\n **Option C \u2014 Mortar & pestle with liquid nitrogen:**\n Grind tissue to a fine powder under LN\u2082. Transfer powder to a tube, let LN\u2082 evaporate, then add Buffer RLT Plus and vortex.\n\n **Option D \u2014 Needle and syringe:**\n Pass lysate through a **20-gauge needle** at least **5\u201310 times** until homogeneous.\n\n4. Centrifuge the lysate at **full speed (\u226512,000 \u00d7 g) for 3 min**, RT.\n5. Carefully transfer the supernatant to a new tube. **Do not disturb the pellet.**\n\n### 2. Bind Genomic DNA\n\n1. Transfer the cleared lysate onto an **AllPrep DNA Mini Spin Column** in a 2 mL collection tube.\n2. Centrifuge at **\u22658,000 \u00d7 g for 30 s**, RT.\n3. **Set aside the DNA column** at 4 \u00b0C (or RT for up to 1 h) \u2014 proceed to RNA purification first.\n4. **Save the flow-through** for RNA isolation.\n\n### 3. Adjust RNA Binding Conditions\n\n1. Add **1 volume of freshly prepared 70% ethanol** to the flow-through (e.g. 600 \u00b5L ethanol to 600 \u00b5L flow-through). For **liver tissue**, use **50% ethanol** instead.\n2. Mix well by pipetting. **Do not centrifuge.** A precipitate may form \u2014 this is normal; proceed immediately.\n\n### 4. Bind and Wash RNA\n\n1. Transfer up to **700 \u00b5L** of the sample onto an **RNeasy Mini Spin Column** in a 2 mL collection tube.\n2. Centrifuge at **\u22658,000 \u00d7 g for 15 s**, RT. Discard flow-through.\n3. Repeat until all sample has passed through the column.\n4. Add **350 \u00b5L Buffer RW1**. Centrifuge at **\u22658,000 \u00d7 g for 15 s**, RT. Discard flow-through.\n\n### 5. On-Column DNase Treatment\n\n1. Pipette **80 \u00b5L DNase Mix** (10 \u00b5L DNase I + 70 \u00b5L Buffer RDD) directly onto the membrane.\n2. Incubate at **RT for 15\u201330 min** (30 min recommended for maximum removal).\n3. Add **350 \u00b5L Buffer RW1**. Centrifuge at **\u22658,000 \u00d7 g for 15 s**, RT. Discard flow-through.\n\n### 6. Wash and Dry RNA Column\n\n1. **Replace the collection tube** with a new 2 mL tube.\n2. Add **500 \u00b5L Buffer RPE** (ethanol added). Centrifuge at **\u22658,000 \u00d7 g for 15 s**, RT. Discard flow-through.\n3. Add another **500 \u00b5L Buffer RPE**. Centrifuge at **\u22658,000 \u00d7 g for 2 min**, RT, to dry the membrane.\n4. Optional: place column in a new 2 mL tube and centrifuge at **full speed for 1 min** to eliminate residual ethanol.\n\n### 7. Elute RNA\n\n1. Place the RNeasy column in a new **1.5 mL collection tube**.\n2. Add **30\u201350 \u00b5L RNase-free water** directly onto the membrane.\n3. Incubate **1\u20135 min** at RT.\n4. Centrifuge at **\u22658,000 \u00d7 g for 1 min**, RT.\n5. Optional: repeat with a second **30 \u00b5L** RNase-free water for maximum yield. Pool eluates if the second elution is \u226530% of the first.\n6. Place RNA on ice immediately.\n\n### 8. Wash Genomic DNA Column\n\n1. Retrieve the AllPrep DNA column from step 2.\n2. Add **500 \u00b5L Buffer AW1**. Centrifuge at **\u22658,000 \u00d7 g for 15 s**, RT. Discard flow-through.\n3. Add **500 \u00b5L Buffer AW2**. Centrifuge at **full speed (\u226514,000 \u00d7 g) for 2 min**, RT, to dry the membrane.\n4. Optional: place column in a new 2 mL tube and centrifuge at **full speed for 1 min** to eliminate residual ethanol.\n\n### 9. Elute Genomic DNA\n\n1. Place the AllPrep DNA column in a new **1.5 mL collection tube**.\n2. Add **50\u2013100 \u00b5L RNase/DNase-free water** (pre-warmed to 70 \u00b0C for improved yield) directly onto the membrane.\n3. Incubate **2 min** at RT.\n4. Centrifuge at **\u22658,000 \u00d7 g for 1 min**, RT.\n5. Optional: repeat with a second **30 \u00b5L** for maximum yield.\n\n### 10. Quantification and Quality Assessment\n\n**RNA:**\n* Measure concentration by Qubit RNA assay or NanoDrop A260.\n* Record A260/280 (expect \u22651.8) and A260/230 (expect \u22651.5) ratios.\n* Assess integrity by Fragment Analyzer, Bioanalyzer, or TapeStation (target RQN/RIN \u22657 for most applications).\n\n**DNA:**\n* Measure concentration by Qubit dsDNA assay or NanoDrop A260.\n* Record A260/280 (expect ~1.8) and A260/230 ratios.\n* Assess fragment size by TapeStation Genomic DNA assay or agarose gel if required.\n\n### 11. Storage\n\n* **RNA:** Keep on ice for immediate use. Store at **\u221280 \u00b0C** for long term.\n* **DNA:** Store at **\u221220 \u00b0C** (short term) or **\u221280 \u00b0C** (long term). Avoid repeated freeze-thaw cycles.\n\n---\n\n## Special Considerations for Muscle, Heart, and Skin Tissues\n\nThese tissues are dense and fibrous, leading to potential column clogging and lower yields.\n\n* Start with **\u226415 mg** (LN\u2082-preserved) or **\u226410 mg** (RNAlater-preserved).\n* **Pre-warm** Buffers RW1 and RPE to **37 \u00b0C** for 30 min before use.\n* Centrifuge lysate **twice** at full speed to minimize cell debris.\n* Load a maximum of **400 \u00b5L** per column pass.\n* If the column clogs: incubate wash solution on column for **5 min at 37 \u00b0C** before centrifuging.\n* Elute with **pre-warmed (37 \u00b0C) water** in two passes (50 \u00b5L + 30 \u00b5L); combine if second elution is \u226530% of first.\n* If yields remain low: process **two 15 mg aliquots separately** and combine eluates.\n\n---\n\n## Acceptance and Rejection Criteria\n\n* Elution volume and expected yield range documented per tissue type.\n* No visible carryover of debris in eluate.\n* Proceed if spectrophotometric and integrity checks meet project-specific thresholds.\n\n## Expected Yields\n\n| Tissue type | DNA yield | RNA yield |\n|---|---|---|\n| Liver (20 mg) | 10\u201330 \u00b5g | 20\u201360 \u00b5g |\n| Brain (20 mg) | 5\u201315 \u00b5g | 5\u201320 \u00b5g |\n| Kidney (20 mg) | 5\u201315 \u00b5g | 10\u201340 \u00b5g |\n| Heart/Muscle (15 mg) | 2\u201310 \u00b5g | 1\u201310 \u00b5g |\n| Spleen (20 mg) | 15\u201340 \u00b5g | 15\u201360 \u00b5g |\n\n## Troubleshooting\n\n| Problem | Possible cause | Solution |\n|---|---|---|\n| Low RNA yield | Incomplete lysis | Increase homogenization time; ensure \u03b2-ME was added to RLT Plus |\n| Low RNA yield | Column overloaded | Reduce input tissue mass |\n| Low DNA yield | Insufficient lysis | Ensure thorough mechanical disruption before loading AllPrep DNA column |\n| Low DNA yield | DNA column dried out | Do not leave DNA column at RT for >1 h |\n| Genomic DNA in RNA | DNase treatment insufficient | Extend incubation to 30 min; verify DNase I activity |\n| RNA in DNA | Carryover of flow-through | Avoid disturbing interface when transferring lysate |\n| Column clogging | Tissue debris | Pre-clear lysate by additional centrifugation; reduce input mass |\n| Low A260/230 | Guanidine salt carryover | Add an extra RPE/AW2 wash step |\n\n## Waste Disposal\n\n* Collect \u03b2-mercaptoethanol and guanidine-containing wastes according to institutional chemical waste procedures.\n* Dispose of biological materials under local biosafety rules.\n* Spin columns with bound nucleic acids are non-hazardous after elution.\n\n## Records to Maintain\n\n* Date, operator, sample ID, tissue type and mass, homogenization method, reagent lot numbers, elution volume(s), DNA and RNA concentrations, purity ratios, integrity assessment, and storage location.\n* Deviations and corrective actions.\n\n---\n\n**End of SOP**"
}
]
},
{
"@id": "#protocol-37",
"@type": "LabProtocol",
"name": "PCR-cDNA Barcoding sequencing library preparation (SQK-PCB111.24)",
"description": "Oxford Nanopore Technologies PCR-cDNA Barcoding kit (SQK-PCS111) for full-length cDNA library preparation from total RNA. Uses template-switching reverse transcription followed by PCR amplification with barcoded primers. Compatible with MinION, GridION and PromethION flow cells (R9.4.1). Superseded by SQK-PCS114 in late 2023.",
"url": {
"@id": "https://community.nanoporetech.com/docs/prepare/library_prep_protocols/pcr-cdna-barcoding-kit-sqk-pcb111-24"
},
"version": "1.0",
"category": "sample_prep",
"step": [
{
"@type": "HowToStep",
"position": 1,
"text": "## Overview & Kit Contents\n\nThis protocol is based on the Oxford Nanopore Technologies **PCR-cDNA Barcoding Kit (SQK-PCB111.24)**, suitable for generating full-length barcoded cDNA sequencing libraries from total RNA.\n\n### Kit components\n- VNP primer, SSP primer (strand-switching primer)\n- Barcoded PCR primers (BC01\u2013BC24)\n- Rapid Adapter (RAP)\n- Elution Buffer (EB)\n\n### Required reagents (user-supplied)\n- Lambda Exonuclease\n- NEBNext Quick Ligation Reaction Buffer\n- T4 DNA Ligase 2M U/ml\n- 10 mM dNTP solution\n- LongAmp Hot Start Taq 2X Master Mix\n- Maxima H Minus Reverse Transcriptase (200 U/ul) with 5x RT Buffer\n- RNaseOUT, 40 U/ul\n- USER (Uracil-Specific Excision Reagent) Enzyme\n- Exonuclease I\n- AMPure XP beads\n\n### Required equipment\n- Thermal cycler, magnetic rack, Qubit fluorometer, vortex mixer, microcentrifuge"
},
{
"@type": "HowToStep",
"position": 2,
"text": "### Input RNA requirements\n\n- **Input**: 1\u201350 ng poly(A)+ mRNA or 50\u2013100 ng total RNA (RIN >= 7 recommended)\n- RNA should be DNase-treated if genomic DNA contamination is suspected\n- Prepare RNA in 7.5 uL nuclease-free water\n- Add 1 uL VNP primer (2 uM) and 1 uL 10 mM dNTPs\n- Incubate at **65 C for 5 min**, then snap-cool on ice for 1 min"
},
{
"@type": "HowToStep",
"position": 3,
"text": "### Reverse transcription and strand switching (~90 min)\n\n1. Prepare RT mix on ice (per reaction):\n - 4 uL 5x RT Buffer\n - 1 uL RNaseOUT (40 U/uL)\n - 1 uL nuclease-free water\n - 2 uL Strand-Switching Primer (SSP, 10 uM)\n - 1 uL Maxima H Minus RT (200 U/uL)\n2. Add 10 uL RT mix to the 9.5 uL RNA/primer/dNTP mix (total 19.5 uL)\n3. Incubate: **42 C for 90 min** (RT + template switching), **85 C for 5 min** (inactivation), hold 4 C\n4. Add 1 uL RNase H, incubate **37 C for 10 min**\n5. Briefly centrifuge and place on ice"
},
{
"@type": "HowToStep",
"position": 4,
"text": "### PCR amplification with barcoded primers (~30 min)\n\n1. Prepare PCR reaction (50 uL total):\n - 20 uL cDNA from RT step\n - 25 uL LongAmp Taq 2X Master Mix\n - 1.5 uL Barcode Primer (BC*XX*, 10 uM)\n - 3.5 uL nuclease-free water\n2. Cycling conditions:\n - **95 C, 3 min** (initial denaturation)\n - **18 cycles**: 95 C 15 s / 62 C 15 s / 65 C 5 min\n - **65 C, 6 min** (final extension)\n - Hold at 4 C\n\n**Note**: 18 cycles is suitable for 50-100 ng total RNA input. Reduce to 14-15 cycles for higher inputs."
},
{
"@type": "HowToStep",
"position": 5,
"text": "### Bead clean-up and quantification\n\n1. Add 40 uL AMPure XP beads (0.8x ratio) to 50 uL PCR product\n2. Incubate at RT for 5 min\n3. Place on magnetic rack, wait for pellet to form\n4. Wash 2x with 200 uL freshly prepared 80% ethanol\n5. Air-dry pellet for ~30 s (do not over-dry)\n6. Elute in 12 uL EB\n7. Quantify with Qubit dsDNA HS assay\n8. Expected yield: 50-500 ng depending on input RNA quality and quantity"
},
{
"@type": "HowToStep",
"position": 6,
"text": "### Pool barcoded libraries and attach Rapid Adapter\n\n1. Pool equimolar amounts of barcoded cDNA libraries (target: 100-200 fmol total)\n2. Adjust volume to 11 uL with EB\n3. Add 1 uL Rapid Adapter (RAP)\n4. Mix gently by flicking, spin down briefly\n5. Incubate at **RT for 5 min**\n6. Library is ready for loading -- **do not vortex** after adapter attachment"
},
{
"@type": "HowToStep",
"position": 7,
"text": "### Flow cell priming and loading\n\n1. Prepare priming mix: 30 uL FCT + 1170 uL FCF\n2. Load 800 uL priming mix via priming port, wait 5 min\n3. Prepare loading mix: 12 uL adapted library + 37.5 uL SB + 25.5 uL LB\n4. Load remaining priming mix, then load 75 uL library via SpotON port\n5. Start sequencing in MinKNOW (Kit: SQK-PCB111.24, enable barcode demultiplexing)"
},
{
"@type": "HowToStep",
"position": 8,
"text": "## References\n\n- Oxford Nanopore Technologies, **\"PCR-cDNA Barcoding Kit (SQK-PCB111.24)\"**.\n- Earlier version: SQK-PCS111. Kit superseded by SQK-PCS114/SQK-PCB114 (V14 chemistry, R10.4.1 flow cells) in late 2023."
}
]
},
{
"@id": "#protocol-44",
"@type": "LabProtocol",
"name": "Fragment Analyzer \u2013 SS NGS Fragment 1-6000 bp",
"description": "Standard sensitivity NGS fragment analysis (1-6000 bp range) on the Fragment Analyzer using the DNF-473 kit. Suitable for QC of NGS libraries, PCR products, and size-selected DNA fragments in the 1-6000 bp range.",
"url": {
"@id": "https://www.agilent.com/store/en_US/Prod-DNF-473-0500/DNF-473-0500"
},
"version": "1.0",
"category": "measurement",
"step": [
{
"@type": "HowToStep",
"position": 1,
"text": "**Method:** `DNF-473-33 - SS NGS Fragment 1-6000bp.mthds`\n\n**Instrument:** Agilent/Advanced Analytical Fragment Analyzer (33 cm capillary array)\n\n**Analysis Mode:** NGS\n\n---\n\n## 1. Document Control\n\n* **SOP ID:** FA-NGS-1-6000bp-33cm\n* **Version:** 1.0\n* **Effective Date:** __________\n* **Prepared by:** __________\n* **Approved by:** __________\n* **Review cycle:** 12 months\n\n## 2. Purpose\n\nTo define the procedure for running standard-sensitivity NGS fragment analysis on the Fragment Analyzer using the **DNF-473-33** method. This method resolves DNA fragments in the **1\u20136,000 bp** range with standard sensitivity, suitable for:\n\n* QC of NGS libraries (amplicon, PCR-cDNA, shotgun)\n* Assessment of size selection efficiency (e.g. AMPure bead selection)\n* Verification of library size distribution prior to sequencing\n* Quantification of library concentration\n\n## 3. Scope\n\nApplies to all operators performing NGS library QC or fragment analysis in the 1\u20136,000 bp range using the DNF-473 kit on a Fragment Analyzer.\n\n## 4. Responsibilities\n\n* **Operator:** Execute this SOP, record run metadata, verify QC, and report issues.\n* **Lab lead:** Ensure training, instrument maintenance, and SOP review.\n\n## 5. Safety\n\n* Wear lab coat, safety glasses, and disposable gloves.\n* Handle gels, buffers, and plates per SDS.\n* Avoid contact with high voltage areas during operation. Only open instrument when software indicates safe.\n* Dispose of consumables as per local regulations.\n\n## 6. References\n\n* Fragment Analyzer user manual.\n* DNF-473 SS NGS Fragment 1-6000bp kit instructions.\n* Local laboratory policies for data retention and equipment maintenance.\n\n## 7. Materials and Equipment\n\n**Instrument and configuration**\n\n* Fragment Analyzer with **33 cm** capillary array.\n* Array serial and usage count recorded before each run.\n\n**Reagents and consumables**\n\n* SS NGS Fragment 1-6000bp gel cartridge compatible with `DNF-473-33`.\n* Buffer reservoirs and rinse solution as per kit.\n* 96-well plate(s) and plate seals or caps.\n* DNA ladder (supplied with kit or compatible sizing standard).\n* Nuclease-free water and low-retention tips.\n\n**Software**\n\n* ProSize 3.0 or later for data analysis and PDF report generation.\n\n## 8. Sample Requirements\n\n* DNA libraries in TE, EB, or nuclease-free water.\n* Recommended input: **0.2\u201350 ng/\u00b5L** (standard sensitivity range).\n* Minimum volume per well: as per plate format (typically 2 \u00b5L sample + diluent).\n* Avoid EDTA >5 mM, high salt, or detergents that interfere with electrokinetic injection.\n* For very low concentration libraries (<1 ng/\u00b5L, e.g. post-bead-selection), results may show concentration but no resolved peaks \u2014 this is expected and the TIM value is still informative.\n\n## 9. Pre-Run Checks\n\n1. Inspect capillary array for leaks, kinks, or salt crystallisation. Record **array serial** and **usage count**.\n2. Confirm instrument details in software:\n * **Device Serial:** record\n * **FA Version:** record\n3. Verify consumables:\n * Correct **gel** loaded and **Gel Selection: Gel 1**.\n * Fresh buffer and rinse trays in correct positions.\n4. Equilibrate reagents to room temperature if required by kit.\n5. Prepare plate map with samples, ladder, controls, and blanks.\n\n## 10. Plate Preparation\n\n1. Thaw and mix reagents gently. Spin down briefly.\n2. Aliquot ladder into designated well (typically position 12 of each row).\n3. Aliquot samples into designated wells per plate map.\n4. Include at least one blank (nuclease-free water) for baseline reference.\n5. Seal the plate. Briefly centrifuge to remove bubbles. Keep sealed until loading.\n\n## 11. Instrument Setup (Software)\n\nSet parameters as follows:\n\n| Parameter | Setting |\n|---|---|\n| Method Name | `DNF-473-33 - SS NGS Fragment 1-6000bp.mthds` |\n| Analysis Mode | `NGS` |\n| Gel Prime | `No` |\n| Full Conditioning | `Yes` (start of day) |\n| Gel Selection | `Gel 1` |\n| Perform Prerun | `5.0 kV` for `30 s` |\n| Rinse | Tray `3`, Row `A`, `# Dips: 2` |\n| Marker 1 | `No` |\n| Sample Injection | `5.0 kV` for `4 s` |\n| Separation | `8.0 kV` for `40.0 min` |\n| # of Capillaries | Per array configuration |\n\n**Note:** The DNF-473 method uses **shorter separation time (40 min)** and **higher separation voltage (8 kV)** compared to the HS Large Fragment method (DNF-464: 60 min, 5 kV), reflecting the smaller target size range.\n\n## 12. Run Procedure\n\n1. Load the sealed sample plate into the instrument.\n2. Confirm tray positions and liquid levels.\n3. In software, confirm the method parameters match Section 11.\n4. Start the run. Observe for the first 2\u20133 minutes for abnormal current, leaks, or errors.\n5. Allow the **40 min** separation to complete.\n\n## 13. Data Analysis (ProSize)\n\n1. Open the completed run in ProSize 3.0.\n2. Verify ladder sizing is correct (expected peaks at kit-specified positions).\n3. For each sample, review:\n * **Electropherogram** \u2014 peak shape, baseline, and resolution\n * **Total Concentration (ng/\u00b5L)** \u2014 integrated from all non-marker peaks\n * **TIM (nmol/L)** \u2014 total integrated molarity\n * **Main peak size (bp)** \u2014 dominant fragment size\n * **Number of peaks** \u2014 indicator of library complexity/purity\n4. Export PDF report containing per-sample peak tables, electropherograms, and summary metrics.\n\n## 14. Interpretation Guide\n\n| Observation | Interpretation | Action |\n|---|---|---|\n| Single clean peak at expected size | Library prep and/or size selection successful | Proceed to pooling/sequencing |\n| Broad peak or multiple peaks | Heterogeneous library or incomplete size selection | Consider re-selection or accept depending on application |\n| No peaks but measurable concentration | Library below detection threshold for sizing | Verify by Qubit; may still be usable if concentration is sufficient |\n| Adapter dimer peak (~100-150 bp) | Incomplete cleanup | Re-clean with AMPure beads at appropriate ratio |\n| No signal | Failed library prep, sample degraded, or loading error | Troubleshoot: check sample, re-run, or re-prep |\n\n### Typical Results for ONT PCR-cDNA Libraries\n\n| Library state | Expected concentration | Expected main peak |\n|---|---|---|\n| Pre-size-selection | 4\u201310 ng/\u00b5L | 1,000\u20132,000 bp (broad) |\n| Post-bead-selection (1.5 kb) | 0.5\u20132 ng/\u00b5L | ~1,480 bp (single peak) |\n| Post-bead-selection (4 kb) | 0.5\u20132 ng/\u00b5L | ~3,500\u20134,500 bp (single peak) |\n\n## 15. Quality Control and Acceptance\n\n* Ladder wells exhibit expected profile and sizing.\n* Baseline is stable with distinct peaks where applicable.\n* Migration time consistent across capillaries.\n* If any criterion fails, document, recondition, and repeat controls before analysing unknowns.\n\n## 16. Troubleshooting\n\n| Problem | Action |\n|---|---|\n| Weak or absent signal | Verify injection settings, sample concentration, plate seal integrity, capillary tips in wells |\n| High noise or drifting baseline | Re-condition array, check buffer freshness and tray cleanliness |\n| Inconsistent sizing | Confirm ladder integrity, plate map accuracy, and temperature stability |\n| Frequent current faults | Inspect for bubbles, low liquid levels, or salt contamination; replace consumables |\n\n## 17. Maintenance\n\n* Perform **Full Conditioning** at the start of the day or as required by kit.\n* Replace gels and buffers per shelf-life and use counts.\n* Track **Array Usage Count** at each run and follow vendor guidance for replacement.\n* Clean trays weekly or after spills. Inspect electrodes and stage area.\n\n## 18. Records to Retain\n\nFor each run record:\n\n* Date, time, and operator\n* Device serial and FA software version\n* Array serial and usage count\n* Method name and key parameters\n* Plate map and sample IDs\n* File path to raw data and PDF reports\n* QC outcomes and any deviations\n\n---\n\n**End of SOP**"
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