title: GSM6297478: HIF-1a/2a, batch2, rep2 [polyA RNA-seq]; Homo sapiens; RNA-Seq
library_construction_protocol: polyA RNA-seq batch1: RNeasy mini plus kit (Qiagen), according to manufacturer instructions polyA RNA-seq batch2: TRIzol RNA extraction (Invitrogen), according to manufacturer instructions, with an additional chloroform extraction step after the phenol:chrloroform extraction polyA RNA-seq batch1: 1 µg of purified RNA was added of ERCC RNA Spike-In Mix 2 (ThermoFisher Scientific # 4456740, 0.5-1 µl of a 1:100 dilution per sample) and the total volume brought to 50 µl with UltraPure Water (ThermoFisher Scientific # 10977-023). mRNA purification, RNA fragmentation, first and second strand cDNA synthesis were performed according to the TruSeq RNA Sample Preparation v2 Kit but using Superscript III for reverse transcription instead of Superscript II (50'C for 50' incubation time). cDNA was purified with AMPure XP beads, and eluted in 27.5 μl of 10mM Tris-HCl pH 7.9, 25 μl of which were transferred to a new tube and subjected to a NEBNext® Ultra™ II DNA Library Prep Kit for Illumina with a few modifications (see linked manuscript) polyA RNA-seq batch2: 5 µg of purified RNA were digested with the Ambion DNA-free DNase treatment (AM1906). 1 µg of digested RNA was subject to poly-A purification and library preparation with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490S) plus the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770S)