6.4. First PCR with Anchor Primers

This step utilizes universal Anchor PCR primers to amplify the target DNA fragments flanked with the Anchor 1 and Anchor 2 sequences generated during the previous Forward Gene-Specific Primer Extension step. The protocol below specified volumes of reagents and Master Mixes for one pooled DNA sample.

1. Prepare the Anchor PCR Master Mix as shown below:
    
   

   Anchor PCR Master Mix Component	Volume per sample, µl
   PCR Buffer, 5X	11
   dNTP Mix	1.1
   Anchor Primer Mix, 10X	11
   Water	31.3
   DNA Polymerase	0.6
   Total	55

2. Gently vortex Master Mix and spin down briefly to collect droplets. Add 50 µl of Anchor PCR Master Mix to each sample test tube from Step 6.3:
    
   

   Component	Volume per sample, µl
   Forward GS Primer Extension DNA (after primer removal step)	53
   Anchor PCR Master Mix (prepared above)	50
   Total	103

3. Mix content by pipetting 3 times and spin down to collect droplets.

*Note: Take the sample test tube from the PCR-free room and perform all follow-up Amplification, Purification, and NGS steps in the location dedicated to PCR work. Never bring amplified library products back to the pre-PCR room.

4. Load the plate in the thermal cycler in a location dedicated to PCR work. Run the following program using the recommended number* of PCR cycles:
    
   

   Temperature	Time	Cycles
   98°C	30 sec	1
   98°C	20 sec	20-26* Note below
   65°C	10 sec
   72°C	45 sec
   72°C	30 sec	1
   4°C	∞	1

*Note: For Pooled sorted single-cell sample run 26 cycles. For scAIR Positive Control RNA sample run 22 cycles. The number of cycles in 1st PCR in many cases needs to be optimized due to differences in transcriptional activity of sorted cells. E.g., activated cells are more transcriptionally active than naïve/dysfunctional cells. For plasma B cells we recommend using 20 cycles. Differences in amplification efficiency between different samples could be also adjusted in 2nd PCR step (see below).

!Stopping Point in procedure: PCR products may be stored at 4°C for a week.