GSM9462058: L3a1 RNA rep2; Homo sapiens; RNA-Seq
The cells were washed with PBS three times, and genomic DNA and total RNA was extracted using AllPrep DNA/RNA mini kit (Qiagen). For DNA/RNA barcode sequencing, mRNA was treated with Turbo DNase to remove contaminating DNA, and reverse transcribed with SuperScript IV (Invitrogen) using a primer containing unique molecular identifiers (UMIs). Resulting cDNA and genomic DNA was split into multiple reactions to reduce PCR jack-potting effects and barcode amplification performed with NEBNext high-fidelity polymerase for three cycles using a forward primer containing P5 adaptor and a sample index and a reverse primer containing P7 adaptor and UMIs. PCR products were purified with AMPure XP beads (Beckman Coulter) to remove primers and concentrate samples. These products underwent a second round of amplification using P5 and P7 primers. All reactions were pooled and run on agarose gels for size selection and submitted for sequencing. For DNA-barcode association, CRE-barcode fragment is amplified with a forward primer upstream from CRE and a reverse primer downstream from barcode, using a plasmid library as a template, followed by AMPure XP beads purification and sequencing.