alias: GSM2466028
title: GSM2466028: Light fraction; control; rep2; Mus musculus; OTHER
library_construction_protocol: RNA was isolated for each fraction using Trizol and Direc-Zol RNA mini-prep kits (Zymo Research). RNA spikes (0.25ng GFP and 1pg Luciferase) that were transcribed (P1300, Promega) and capped (S1407, NEB) in-vitro using T7 RNA Polymerase were added into each fraction. Equivalent RNA volume was taken from each fraction for the library preparation for TSSs sequencing using the CapSeq method (with modifications) as previously described (Gu et al. 2012). Specifically, the RNA samples were treated with Terminator 5´-Phosphate-Dependent Exonuclease (TER51020, Epicenter). Then, reaction volume was enlarged and CIP (M0290, NEB) was added to dephosphorylate non-mRNA RNAs (tRNA and 5S rRNA). DNaseI was added in this step. In order to remove mRNA 5’Cap, the samples were treated with TAP (T19050, Epicenter) and then RNA linker was ligated at the 5’end of the formerly capped mRNA by T4 RNA ligase (M0204, NEB). cDNA was prepared from each RNA sample by using hexamer random primers linked to illumina 3’ Rd2 seq primer, index (unique barcode for each sample), 4nt-long UMI and P7 illumina adaptor. To increase the cDNA quantity, a linear PCR amplification (second strand synthesis) was performed with forward primer containing the ligated linker sequence and carry P5 illumina adaptor. Final library amplification PCR step was performed after calibrating the numbers of PCR cycles. Size selection with magnetic beads (Ampure XP, according to the manufacturer guidelines) was performed after cDNA for removal of fragments <~150 nt and after linear amplification and final PCR steps, in which sizes of >200 bp and < 500 bp were selected. The deep sequencing was performed with HiSeq High-Throughput Sequencing System.
identifiers.namespace: GEO
identifiers.id: GSM2466028