cDNA from Jurkat replicate 2 of PIBv1 MPRA library, sequencing lane 4
MPRA library with GFP ORFs (mpra:gfp) was transfected into 5 biological replicates of K562 cells and 4 biological replicates of Jurkat cells using the Neon Transfection System 100L Kit. For K562, we transfected 90M cells with 10 g plasmid per 10M cells using 3 pulses of 1450V for 10ms per replicate. For Jurkat, we transfected 160M cells with 10 g plasmid per 10M cells using 3 pulses of 1350V for 10ms per replicate. Twenty-four hours after transfection, cells were harvested, rinsed with PBS, and collected by centrifugation, then after adding RLT buffer, DTT, and homogenizing, cell pellets were frozen at -80C. RNA was then extracted from frozen cell pellets using the Qiagen RNeasy Maxi kit, treated with DNase, and then GFP transcripts were captured using a mixture of 3 GFP-specific biotinylated primers (GFP_BiotinCapture_1_120, GFP_BiotinCapture_2_123, and GFP_BiotinCapture_3_126) with Dynabeads MyOne Streptavidin C1 beads. Following a second DNase treatment, cDNA was synthesized using SuperScript III, quantified by qPCR, and then biological replicates were diluted to approximately the same concentration across replicates. Normalized cDNA was then subjected to a first round of PCR (9 cycles for K562 replicates, 16 cycles for Jurkat replicates) with custom primers (MPRA_Illumina_GFP_F_v2_781 and Ilmn_P5_1stPCR_v2_782) to amplify barcodes linked to each GFP mRNA. Each resulting cDNA library went through a second round of PCR (6 cycles for all) in parallel alongside aliquots of diluted mpra:gfp library (one aliquot per cell type per replicate) in separate reactions in order to add Illumina sequencing adapters. The resulting tag-sequencing cDNA and mpra:gfp paired libraries were then all pooled together and sequenced on 4 lanes of Illumina NextSeq 2000 using 1x75 bp reads with dual 8 bp i5 and i7 index reads.
SUB16055622
PIBv1_Jurkat_rep2_L004