GSM2871548: Differentiated Ctrl sgRNA 3; Homo sapiens; RNA-Seq
Differentiated and undifferentiated U937 cells were seeded at a density of 50,000 cells per well in a 24-well plate. Two days later, cells were lysed in RLT buffer and RNA was isolated using the RNeasy Micro Kit (Qiagen). cDNA libraries were prepared and adapted for sequencing using a modified Smart-seq2 protocol (Picelli et al., 2014). In short, 10 ng of purified RNA was mixed with RNase Inhibitor, Oligo-dT30 primer, and dNTPs and heat-denatured before addition to a SuperScriptII template-switching reverse transcription reaction mixture. cDNA was amplified in a 12-cycle KAPA HiFi HotStart PCR reaction and purified using Ampure XP SPRI beads. 0.5 ng of cDNA was fragmented for sequencing using the Nextera XT DNA sample preparation kit. After tagmentaion and amplification of adapter-ligated, indexed fragments, DNA was purified using Ampure XP SPRI beads and sequenced on a NextSeq 550 (Illumina) to obtain 75 bp paired-end reads.
GEO Accession
GSM2871548