title: GSM6862261: mpra_N-D2_3; Homo sapiens; OTHER
library_construction_protocol: To perform the MPRA in cell lines, an MPRA lentiviral library titration was performed to determine the volume of concentrated lentivirus needed to achieve a high infection rate without negatively affecting cell growth. Titration was also performed to test the optimal concentration of blastocidin S HCl needed for a 48 hour selection. For each sample, 10 million cells were transduced with 700µL of the MPRA lentiviral library and 5 µg/mL polybrene. Cells were plated into two 10cm plates (5 million cells/plate) and cultured overnight. Approximately 24 hours post transduction, cells were lifted and plated into two 15cm plates in media supplemented with blasticidin S HCl. Non-transduced cells were fully selected within 48 hours. After 72 hours of drug selection, cells were collected and lysed in Qiagen buffer RLT Plus containing 2-mercaptoethanol. Transductions were performed in triplicate. Lysates were frozen at -80°C prior to performing RNA isolation using a Qiagen RNeasy Plus Mini Kit (Qiagen cat. no. 74136). Both the plasmid library and cDNA libraries were sequenced. The plasmid library was sequenced to assess oligo frequencies. Briefly, Illumina adaptors were PCRed onto the plasmid libraries, upstream and downstream of the oligo inserts. Final libraries concentrations were assessed by Kapa Library Quantification Kit (Roche) and sequenced on an Illumina MiSeq instrument using 30 cycles for read 1 (barcode) and 115 cycles for read 2 (genomic instance). Paired-end sequencing was used to assess template switching.  cDNA libraries were synthesized from total RNA.  Briefly, SuperScript IV was used to synthesize cDNA with a sample specific primer that annealed to the MPRA mRNA transcript. A 12 bp random sequence was included in the primer to serve as a transcript UMI. cDNA synthesis reactions were size selected using SPRIselect beads (Beckman) to remove the cDNA synthesis oligo. Partial Illumina sequencing adaptors were added using PrimeSTAR Max DNA Polymerase (Takara) in a PCR1 consisting of five cycles. Full adapters and sample indices were added in a PCR2 and monitored by qPCR on a Stratagene MX3005P machine. Amplification was terminated while qPCR curves were still in the linear phase to avoid library-over amplification and minimize library skew. The final library was gel purified in a 2% agarose gel. Library concentrations were determined using a Kapa Library Quantification Kit (Roche) and on an Agilent Bioanalyzer. Average molarity was used to equally mix a final cDNA library. Libraries were sequenced on an Illumina MiSeq instrument to ensure each sample had adequate coverage. Deeper sequencing runs were performed on an Illumina Novaseq 6000 SP flow cell (Novogene) with 50 read 1 cycles, 8 index 1 cycles, and 20 index 2 cycles.