title: GSM8794540: OS-RC-2, sgHIF2a, Rep3; Homo sapiens; RNA-Seq
library_construction_protocol: OS-RC-2 cells were homogenized with QIAshredder columns (Qiagen 79654), and total RNA was extracted from homogenized cell lysates using the RNeasy Mini Kit (Qiagen 74106) according to manufacturer's protocol. The RNA was quantified on a NanoDrop spectrophotometer (Thermo Scientific ND-8000-GL). Ribosomal RNA (rRNA)-depleted libraries were prepared using KAPA RNA HyperPrep Kit with RiboErase (Roche) from 100 ng of total RNA according to manufacturer's protocol. Briefly, mRNA or non-coding RNA were enriched and then purified using KAPA pure beads and fragmented at 94°C for 8 min. 1st strand and 2nd strand were subsequently synthesized. cDNA fragments were end-repaired and adenylated at the 3′ ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment with 14 cycles of PCR post-adapter ligation on a Beckman Coulter Biomek i7. Finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced with paired-end 150 bp reads on an Illumina NovaSeq 6000 at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.