GSM2176372: Wt2R; 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. 30 µg of total RNA was fractionated into a long (<200 nt) and a small RNA fraction (<200 nt) by a modified protocol for small RNA cloning (W. Gu and D. Conte) using mirVana Isolation Kit buffers. Briefly, 400 μl of Lysis/Binding Buffer was mixed with 48μl of Homogenate Additive and 80 µl of total RNA. The mix was incubated at RT for exactly 5 min. 1/3rd volume of Ethanol was added and mixed by inversion. This was incubated at RT for exactly 20 min to increase yield. 10 μg GlycoBlue was added and spun at 21 °C and 2,500 x g for 8 min to pellet the long fraction. The supernatant containing the small fraction was carefully transferred to a fresh tube and precipitated with isopropanol and 15 μg GlycoBlue at -80 °C for at least 10 min. rRNA depletion was carried out on the small fraction and on the half amount of the long fractions according to the RiboMinus Eukaryote System v2 (Ambion) protocol. The other half amount of the long fraction was further processed and sequenced as Ribo+ sample. rRNA-depleted samples were concentrated using ethanol precipiptation. The long fractions 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
GSM2176372