GSM3016080: Pt2A N2 DNA rep1; Pan troglodytes; OTHER
The cells were washed with PBS three times, and genomic DNA and total RNA was extracted using AllPrep DNA/RNA mini kit (Qiagen). Messenger RNA (mRNA) was purified from the total RNA using Oligotex mRNA mini kit (Qiagen) and treated with Turbo DNase to remove contaminating DNA. mRNA was reverse transcribed with SuperScript II (Invitrogen) using a primer downstream from the barcode. Resulting cDNA was split into multiple reactions to reduce PCR jack-potting effects and cDNA amplification performed with Kapa Robust polymerase for three cycles, incorporating unique molecular identifiers (UMIs) of 10 bp length. PCR products were cleaned with AMPure XP beads (Beckman Coulter) to remove primers and concentrate samples. These products underwent a second round of amplification in 8 reactions per replicate for 15 cycles, with a reverse primer containing only P7. All reactions were pooled and run on agarose gels for size selection and submitted for sequencing. For DNA, each replicate was amplified for 3 cycles with UMI-incorporating primers, just as the RNA. First round products were cleaned up with AMPure XP beads, and amplified in split reactions, each for 20 cycles. Again, reactions were pooled and gel-purified and sequenced.