GSM9540838: d3 RNA-seq R2; Mus musculus; RNA-Seq
RNA was extracted from mESCs and NIH/3T3 fibroblasts by the standard TRIzol extraction procedure (Thermo Fisher). rDNase (Qiagen) digestion was carried out, and purification was performed by spin column (Qiagen) or by phenol: chloroform extraction and ethanol precipitation.  4SU labelling was carried out as detailed previously, with a few modifications. Cell culture media was removed, filtered, and 1 mM 4SU (Glentham Life Sciences) was added. The cells were returned to the incubator and labelled with 4SU for 15 minutes before medium aspiration and TRIzol addition. 1/5 volume of chloroform was added, followed by isopropanol precipitation. The pellet was then washed with 85% ethanol, dried, and resuspended. RNA integrity was checked for a selection of samples using Bioanalyzer (Agilent Technologies). 4SU incorporation was assessed using a slot blot. RNA clean-up was performed by phenol/chloroform/isoamyl alcohol, and precipitation by isopropanol. The pellet was washed with 85% ethanol, dried, and resuspended. The TT-TSS-seq and Poly(A)-TSS-seq methods were previously described in detail. In short, total RNA was dephosphorylated with quickCIP (NEB, 1.2 U/µg RNA. RNA was extracted using phenol/chloroform/isoamyl alcohol. RNA was treated with mRNA decapping enzyme (NEB, 0.7 U/µg RNA) followed by purification by phenol/chloroform/isoamyl alcohol. RNA and the 5' adaptor ligation was performed T4 RNA ligase I (30 U). RNAClean XP (1.8X ratio, Beckman Coulter) was used to remove unligated adaptors, according to the manufacturer's instructions. Poly(A)+ RNA was enriched using Dynabeads Oligo(dT)25 (Thermo Fisher), or 4SU+ RNA was purified as detailed in the TT-chem-seq protocol. RNA was fragmented, and 3' ends were fixed by quickCIP treatment (NEB, 30 U). Pre-adenylated 3' adaptor (2 µM) were denatured and ligation was carried out by T4 Rnl2tr (NEB,200 U). Reverse transcription was carried out by SuperScript III (2 µl, Invitrogen). AMPure XP beads (Agencourt, Beckman Coulter) were used to clean up the cDNA. cDNA pre-amplification was performed with i5_s and i7_s (300 nM each) primers and Phusion HF PCR Mastermix (Thermo Fisher). 6 PCR cycles were carried. ProNex beads (Promega) with a 1:2.95 ratio to sample were used to remove sequences less than 55 nt (including primer dimers). Final amplification was carried out with NEBNext i50 and i70 primers (500 nM each) and Phusion HF PCR Mastermix, for 8 cycles. Amplification was checked by using Novex 6% TBE (Tris/Borate/EDTA) gel and staining with SYBR Green.For RNA-seq, libraries were prepared using NEB ultra II, with poly(A)+ purification, according to the manufacturer's protocol. Sequencing was performed on NovaSeq 6000 (Illumina) using 100 bp paired-end reads.  For TTchem-seq, at least 10 ng of eluted RNA was used for library preparation with NEBNext Ultra II Directional Poly(A) mRNA, according to the manufacturer's protocol for fragmented RNA. Libraries were sequenced using NovaSeq 6000 (Illumina) as 100 bp paired-end reads.