GSM2304837: rep2_drug_DMSO_PCR2 (rep2); Homo sapiens; OTHER
Genomic DNA and total RNA from the same infected cell pool were extracted by the AllPrep DNA/RNA Mini Kit. Total RNA was further proceeded to purify mRNA by Oligotex mRNA Mini Kit. Libraries used for mapping HIV integrations were prepared by inverse PCR. Briefly, 3 µg DNA were harvested and digested by the restriction enzyme BplI. Fragments were blunt-end self-ligated in the total volume 1 mL at 16℃ overnight. The pellet was dissolved in 84 µL distilled water for plasmid-safe DNase treatment. DNase-treated samples were cleaned up by the QIAquick PCR purification kit. Samples were eluted in 20 µL EB buffer. 8 µL eluted product were used for two rounds of nested PCR. PCR products with different indexes were pooled together in the final concentration of 4 nM for sequencing 50 bp paired end on a Illumina NextSeq sequencer. RNA and DNA libraries were prepared by RT-PCR and regular PCR, respectively, with the same primers. RNA and DNA libraries were sequenced 50 bp single read by on a Illumina NextSeq sequencer.