GSM8223873: Ark1 cells, siGATA2, replicate 1; Homo sapiens; RNA-Seq
Total RNA extraction using TRIZOL Each RNA was assayed on NanoDrop One Spectrophotometer and Agilent 4200Tapestaion, to assess purity and integrity.   RNA samples that met Illumina's “TruSeq Stranded mRNA Reference Guide (Document# 1000000040498 v00, October 2017) quality criteria were prepared for sequencing.  Briefly, 1ug total RNA sample was enriched for mRNA by oligo (dT) selection followed by fragmentation using divalent cations under elevated temperature.  Double-stranded cDNA was synthesized using SuperScript II RT (Invitrogen, Carlsbad, California) and random primers for first strand cDNA synthesis followed by second strand synthesis using DNA Polymerase I and RNAse H for removal of mRNA.  Double-stranded cDNA was purified with SPRI beads (Beckman Coulter Genomics, CA), followed by incubation with Klenow DNA Polymerase and Adenine to add an 'A' base (Adenine) to the 3' end of the blunt phosphorylated DNA fragments.  DNA fragments were  ligated to Illumina unique molecular index (UMI) adapters, which have a single 'T' base (Thymine) overhang at their 3'end.  Following ligation, cDNA was purified with SPRI beads, and PCR-amplified (11 cycles) with PhusionTM DNA Polymerase using Illumina's genomic DNA primer set and purified with SPRI beads.   Quality and quantity of the finished libraries were assayed on Agilent 4200Tapestation using D1000 Screentape and  Qubit HS Quantification Kit (Invitrogen, Carlsbad, CA), respectively.