title: GSM2221118: Wild-type_integrase_HepG2_RNA_rep3; Homo sapiens; OTHER
library_construction_protocol: 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. For each RNA replicate, 3x500ng was reverse transcribed with SuperscriptII (Invitrogen 18064-014) using a primer downstream of the barcode, which contained a sample index and a P7 Illumina adaptor sequence. The resulting cDNA was pooled and split into 24 reactions, amplified with Kapa Robust polymerase for 3 cycles using this same reverse primer paired with a forward primer complementary to the 3’ end of EGFP with a P5 adaptor sequence (BARCODE_lentiF_v4.1, Table S3). The implemented two-round PCR set-up is supposed to reduce PCR jack-potting effects and allows for incorporating unique molecular identifiers (UMIs), which could be used to correct for other PCR biases in future experiments. PCR products are then cleaned up with AMPure XP beads (Beckman Coulter) to remove the primers and concentrate the products. 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 at this point, run on an agarose gel for size-selection, and submitted for sequencing. For the DNA replicates, 16x500ng of each replicate was amplified for 3 cycles just as the RNA. First round products were cleaned up with AMPure XP beads, and amplified for another 16 reactions, each for 20 cycles. Reactions were pooled, gel-purified, and sequenced.
identifiers.namespace: GEO
identifiers.id: GSM2221118