GSM2176383: Tet TKO, mRNA, sample 3; Mus musculus; OTHER
RNA extraction was performed according to the Invitrogen TRIzol RNA isolation protocol (Ambion). The nucleic acid concentrations and purity were analyzed on the NanoDrop ND-1000 Spectrophotometer. RNA integrity was measured on a Bioanalyzer (Agilent) or TapeStation (Agilent). Samples were stored at -80 °C. Poly(A)+ RNA was isolated from 500 µg total RNA by two rounds of purification using polyA Spin mRNA Isolation kit (New England Biolabs). Poly(A)+ RNA were further processed with the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs). This was carried out as described in the manual. 3 min of fragmentation at 94 °C has been established to lead to a peak at approximately 250 nt, appropriate for the final 100 bp paired-end sequencing. The fragmented RNA was precipitated using ethanol with 20 μg GlycoBlue at -80 °C for at least 10 min.   TURBO DNase (Ambion) was applied to each sample according to the protocol in a total volume of 20 μl. This volume was immediately applied to the EZ RNA Methylation Kit (Zymo Research), following the manufacturer’s manual. As a final step before library preparation, a stepwise RNA end repair was carried out using T4 polynucleotide kinase (TaKaRa). 3’-Dephosphorylation and 5’-phosphorylation (using 10 mM dATP) were carried out at 37°C for 20 min each. RNA was further purified by a phenol/chloroform extraction and ethanol precipitation.  Library preparation was done according to the NEBNext Small RNA Library Prep Set protocol. Following adaptor ligation and cDNA synthesis, cDNA was amplified with 12 cycles of PCR and purified using the QIAquick PCR Purification Kit (Qiagen). The libraries were further size selected on a 6% polyacrylamide gel. Compatible barcodes were selected and samples were pooled in equimolar ratios on multiple lanes on an Illumina HiSeq 2000 platform. A 100 bp paired-end sequencing approach was used.
GEO Accession
GSM2176383