GSM8305416: R1-scMPRA; Homo sapiens; RNA-Seq
Cells were fixed using Parse Bio Evercode Fixation kit Parse Bio Evercode Mini v2 kit with the following modifications to the kit protocol for cDNA amplification steps to prepare an additional sequencing library enriching for MPRA amplicons. First, we spiked-in a MPRA amplicon targeting primer GB39 to initial cDNA amplification reaction at the final concentration of 400nM. We performed a second PCR, starting from 50ng of the cDNA from the initial amplification round, using GB42/GB43, with the following conditions: 0.02U/uL NEB Q5 HotStart Polymerase, 1x NEB Q5 Reaction Buffer, 300nM each primers, 20uM dNTPs, 1x Biotium EvaGreen dye; 98°C 20s - 67°C 10s - 72°C 30s. The cycling was stopped after the exponential phase, and the reaction products were double size-selected using Roche KAPA Pure beads at 0.8x-1.5x. Index PCR was performed according to the kit protocol alongside the whole transcriptome library. The final indexed libraries were quantified using KAPA Library Quantification Kit (Roche 7960140001) for pooling and sequenced at 2x150 cycles on NovaSeq X Plus by Novogene.