The Evercode Whole Transcriptome v3 workflows can be completed in two days, as outlined in the figure below. This timing applies to WT Mini v3, WT v3, and WT Mega v3 products.
The protocol also includes 6 optional safe stopping points to allow researchers flexibility in how they complete their experiment. The table below includes further details on timing and indicates the safe stopping points throughout the workflow.
- SECTION | DESCRIPTION | TIME | HANDS-ON-TIME | SAFE STOPPING POINTS
- 1 | Section 1: In Situ Cell/Nuclei Barcoding
- 1.1 Set up and Sample Counting | Variable (30-90 min) |
Variable
(30-90 min)
- 1.2 Barcoding Round 1 | 75 min | 45 min
- 1.3 Barcoding Round 2 | 60 min | 45 min
- 1.4 Barcoding Round 3 | 30 min | 15 min
- 1.5 Lysis and Sublibrary Generation | 60 min | 45 min | -80°C ≤ 6 months
- 2 | Section 2: cDNA Capture and Amplification
- 2.1 cDNA Capture | 60 min | 30 min
- 2.2 cDNA Template Switch | 120 min | 30 min | 4°C ≤ 18 hrs
- 2.3 cDNA Amplification | 90 min | 15 min | 4°C ≤ 18 hrs in the thermocycler
- 2.4 Post-Amplification Purification | 30 min | 30 min
- 2.5 cDNA Quantification | 30 min | 30 min | 4°C ≤ 48 hrs or -20°C ≤ 3 months
- 3 | Section 3: Sequencing Library Preparation
- 3.1 Fragmentation and End Prep | 60 min | 30 min
- 3.2 Fragmentation and End Prep Size Selection | 30 min | 30 min | 4°C ≤ 18 hrs or -20°C ≤ 2 weeks
- 3.3 Adapter Ligation | 30 min | 15 min
- 3.4 Post-Ligation Purification | 30 min | 30 min
- 3.5 Barcoding Round 4 | 45 min | 15 min | 4°C ≤ 18 hrs in the thermocycler
- 3.6 Post-Barcoding Round 4 Size Selection | 30 min | 30 min
- 3.7 Sequencing Library Quantification | 30 min | 30 min | 20°C ≤ 3 months
Please see the Evercode Whole Transcriptome v3 user manuals for additional information.