title: GSM3935999: 4765_2_PC3_ASNase_TTAGGC_S32_R1_001.fastq.gz; Homo sapiens; RNA-Seq
library_construction_protocol: CRISPR-Cas9 screen: Genomic DNA was isolated using the phenol-chloroform extraction protocol and sgRNAs were amplified using a two-step PCR protocol for next-generation sequencing. RNA-seq: Total RNA was isolated using Trisure reagent (Bioline) following the manufacturer's instructions. Briefly, cells were washed twice with PBS and 1 ml Trisure was added for homogenization. After centrifuge, the aqueous phase was transferred to a new tube and mixed with cold isopropyl alcohol for RNA precipitation by centrifuging at 4 °C for 1 hour. RNA pellet was washed twice with 75% ethanol and finally dissolved in RNase-free water. RNA-seq: Stranded-specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina) following the manufacturer's instructions. Briefly, 2 mg of total RNA was polyA-selected using oligo-dT beads and the RNA was fragmented, random primed and reverse transcribed using SuperScript II Reverse Transcriptase kit (Invitrogen). Second strand complementary DNA was then synthesized, 3'-adenylated and ligated to Illumina sequencing adapters, and subsequently amplified by 12 cycles of PCR. The sequencing libraries were analyzed on a 2100 Bioanalyzer using a 7500 chip (Agilent) and pooled equimolarly into a 30 nM multiplex sequencing pool.
attributes.tag: GEO Accession
attributes.value: GSM3935999