6.5. Second PCR with Indexed Primers

This step adds a dual unique DNA/RNA UDP index combination to each 1st PCR Product generated in the previous step, as well as universal flanking P5 and P7 sequences needed for cluster formation on the Illumina NGS flow cell (Outline).

The Dual Index Primer Set Plate is sold separately and contains a unique combination of Forward and Reverse DNA/RNA UDP index primers in each of 96 wells. Each well contains 20 μL of unique index primer combination (10X concentration), which is enough to run x4 single-cell plates (5 μl per 50 μl reaction). One unique index primer combination should be used for each sample (triplicate samples have different indexes). For a small number of samples (e.g., 2-3 plates with sorted cells), consider running the 2nd PCR step in triplicate using different index primers to avoid low-complexity index sequencing in the NGS step. Always record the well coordinates of index primers used for each sample to avoid problems in the NGS sequence deconvolution step. Prior to use, thaw the Dual Index Primer Set Plate, briefly centrifuge the plate to collect droplets at the bottom of the wells, remove the seal slowly (to prevent cross-contamination), and aliquot 5 μl for each sample (45 μl) as described below. Seal the plate with a new sealing film and store at +4 °C (for immediate use) or at -20 °C (for prolonged storage). See Appendix. List of Dual DNA/RNA UDP Indexes for the sequences of each Forward and Reverse Index combination.

Arrangement of the index pairs in the supplied Index Primer Plate.

1. Prepare enough of the 2nd PCR Master Mix, following the formulation below for all samples and controls, plus 5% extra volume of all components:

**Note: If you are using a multichannel pipette, aliquot the master mix in a tube strip to minimize pipetting steps.

2. Gently vortex the 2nd PCR Master Mix, then briefly spin down to collect droplets. Remove the plate seal from the Index Primer Master Plate.

3. Aliquot 45 µl of the 2nd PCR Master Mix into selected wells of the new 96-well plate and add 5 μl of unique index primer combination in each well from the Dual Index Primer Set Plate. For each sample aliquot 2nd PCR Mix in 3 different wells (e.g. A1, A2, A3 for sample 1 and A4, A5, A6 for sample 2). To avoid index-to-index contamination, add index primers from the Index Primer Plate using a new tip for each well. Seal the Index Primer Plate with a new sealing film and store at +4 °C for up to 3 months for future use.

4. Spin down, then remove the seal from the 1st PCR Plate. Transfer 2 µl of the 1st PCR products to each of the three Index Primer PCR reactions allocated for each sample (e.g., aliquot sample 1 in wells A1, A2, A3 and sample 2 in wells A4, A5, A6) prepared in step 3. We recommend that you immediately record the Sample name and well number/index number (e.g., Sample 1 in wells A1, A2, A3 and sample 2 in wells A4, A5, A6 ) for all the samples, including the positive control. This will help minimize mistakes in the NGS deconvolution step.

4. Seal the plate (or portion of the plate) with new adhesive film, and spin it down to collect droplets. Load the plate in the thermal cycler, and run the following program:
    
   

   Temperature	Time	Cycles
   98°C	30 sec	1
   98°C	20 sec	8
   65°C	10 sec
   72°C	45 sec
   72°C	30 sec	1
   4°C	∞	1

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