Evercode Single Index PCR
Parse Biosciences, Elisabeth Rebboah
Abstract
This protocol describes the single-index PCR procedure for Parse Biosciences Evercode WT and WT Mega v2 kits. Each subpool is barcoded with a single Illumina index on the 3' end of the cDNA library. This acts as the fourth "round" of cell barcoding and must be included in the final cell ID/barcode in order to ensure unique barcodes across subpools within an experiment. The numerical ID and sequence of the Illumina barcode used for each subpool are recorded in experiment metadata and used downstream for subpool demultiplexing after the sequencing run.
Steps
Appendix D2: Sublibrary Single Index PCR
If using unique dual indexes (UDIs) instead of sublibrary single index primers for indexing, see Section 3.5. Otherwise, replace the entirety of Section 3.5 with the following steps.
Multiple thermocyclers may be needed for this section depending on the amount of cDNA added to each sublibrary during the fragmentation reaction. Refer to step next page to determine how many thermocyclers are needed.
Using a new 1.5 mL tube, combine the Universal Index Primer and Index Primer Mi x to make the Sublibrary Amplification Mi x. Mix well by pipetting and store on ice.
| A | B | C | D | E | F | G | H | I | J |
|---|---|---|---|---|---|---|---|---|---|
| # Sublibraries | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 16 |
| Index PCR Mix | 27.5 | 55 | 82.5 | 110 | 137.5 | 165 | 192.5 | 220 | 440 |
| Universal Index Primer | 2.2 | 4.4 | 6.6 | 8.8 | 11 | 13.2 | 15.4 | 17.6 | 35.2 |
| Total | 29.7 | 59.4 | 89.1 | 118.8 | 148.5 | 178.2 | 207.9 | 237.6 | 465.2 |
Add 2 μL of different index primers to each sublibrary ensuring that no two sublibraries contain the same sublibrary index primer. Make sure to record which sublibrary contains which index primer.
Add 27 μL Sublibrary Amplification Mix to the 23 μL sublibrary from the previous step. Pipette up and down 10x with the pipette set to 27 μL to ensure proper mixing, followed by brief centrifugation (~2 sec).
Place the samples(s) into a thermocycler and run the program below. The number of cycles (X) should be adjusted based on the amount of cDNA added to the fragmentation reaction.
| A | B | C |
|---|---|---|
| ~30 min | 105C | 50 uL |
Sublibrary Index Amplification Overview
| A | B | C |
|---|---|---|
| 1 | 3 min | 95C |
| 2 | 20 sec | 98C |
| 3 | 20 sec | 67C |
| 4 | 1 min, then go to step 2, repeat X-1 times (X cycles total) | 72C |
| 5 | 5 min | 72C |
| 6 | Hold | 4C |
Sublibrary Index Amplification
| A | B | C | D | E | F | G |
|---|---|---|---|---|---|---|
| cDNA in Fragmentation (ng) | 10-24 | 25-49 | 50-99 | 100-299 | 300-999 | 1,000+ |
| Total PCR cycles required (X) | 13 | 12 | 11 | 10 | 9 | 7 |
PCR Cycles based on cDNA in Fragmentation
Note: cDNA concentration was recorded in step 2.5.18, and 10 μL from each sublibrary should have been added into the fragmentation reaction (step 3.1.2).
Sublibraries can be stored at this point at 4°C overnight. If you wish to continue, proceed directly to Section 3.6: Post-Amplification Double-Sided Size Selection.
[STOPPING POINT]
