title: GSM6280723: RNA-seq.neuron.WT_15days_rep1; Mus musculus; RNA-Seq
library_construction_protocol: RNA-seq: Total RNA was extracted using TRI-reagent and purified using the Direct-zol RNA MiniPrep Kit (Zymo). For TOP1-ChIP, mESCs were harvested and double-crosslinked in suspension with 2mM DSG in PBS for 45 minutes at room temperature, washed with PBS thrice, and incubated with 1% formaldehyde (FA) in PBS for 10 minutes at room temperature. Crosslinking was quenched by addition of glycine to final concentration of 500mM and incubated at room temperature for 5 min. Cells were washed in PBS and snap frozen for storage at -80degC until ChIP was performed. For RNAP2(S2P) ChIPs, mESCs were subjected to crosslinking in 1% FA for 10 minutes at room temperature. Fixed cells were then subjected to standard ChIP lysis protocol to release chromatin. Released chromatin was sonicated to obtain fragments between 100-300bp, pre-cleared and used for IP with antibody-Dynabead complex. Captured fragments were subjected to several rounds of washes before elution with SDS buffer. DNA was purified using SPRI method. RNA-seq in 293T and mESC: Equal volumes of RNA preparations (extracted from equal number of cells) from each sample were each mixed with 1 microl of 1:100 diluted Mix 1 of ERCC RNA Spike-In (Life Technologies Cat.# 4456739). The mixture is subjected to ribosomal RNA depletion using NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (NEB Cat# E6310) and library construction using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB Cat# E7760), according to manufacturer's protocol. RNA-seq in neurons: Equal amounts of RNA were directly used in rRNA depletion and library construction. ChIP-seq: Eluted DNA and DNA from relevant input materials was purified and made into libraries using NEBNext Ultra II DNA kit (NEB Cat #: E7645). For both RNA-seq and ChIP-seq libraries, average fragment size was determined on Agilent High Sensitivity DNA and D1000 Screentape system (Agilent Cat #:5067-4626, 5067-5582), quantified using Qubit High Sensitivity DNA system (Life Tech Cat# Q32854), and pooled in equimolar. Pooled libraries were sequenced on the Illumina NextSeq or HiSeq platform.