GSM6482065: RNA-seq_PAF1AID-DLD1_iPAF1C_Rep2; Homo sapiens; RNA-Seq
Extraction of total RNA was performed using RNeasy Mini kit (Qiagen, Cat# 74106). Briefly, 1million of DLD1_PAF1_AID cells were plated in a 6-well plate 24 hrs prior to treatment with 20μM iPAF1C versus DMSO or 500 μM auxin versus non-treated cells. ChIP-seq: ChIP-seq was performed according to a previously published protocol (Lee et al., 2006). For RNA Pol II, and PAF1 ChIP-seq, the chromatin was sonicated using Covaris E220 for 4 min using the following sonication conditions: 10% duty cycle, 140 peak intensity power, 200 cycles per burst. Successively, pulldown of chromatin was carried out overnight at 4°C using the specific antibodies (Rpb1 NTD (D8L4Y) rabbit mAb for RNA Pol II and PAF1 (D9G9X) Rabbit mAb). DNA libraries were prepared by the HTP Library Preparation Kit for Illumina (KAPA). RNA-seq: Extraction of total RNA was performed using RNeasy Mini kit (Qiagen, Cat# 74106). Briefly, 1million of DLD1_PAF1_AID cells were plated in a 6-well plate 24 hrs prior to treatment with 20μM iPAF1C versus DMSO or 500 μM auxin versus non-treated cells. For performing RNA-seq polyadenylated RNA was isolated using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, Cat# E7490L). This isolated poly(A)+ RNA was used to prepare libraries using NEBNext Ultra II Directional RNA Library Prep Kit (NEB, Cat# E7760L). PRO-seq: PRO-seq was performed according to the previously published protocol with minor modifications (Mahat et al., 2016a). Reverse transcription was performed with SuperScript III (ThermoFisher, Cat#18080044). cDNA was amplified using Phusion Hot Start II DNA polymerase (ThermoFisher, Cat#F549). DNA Libraries were purified using Beckman Coulter AMpure XP (Fisher Scientific, Cat#NC9959336).