GSM2627383: UN rep2; Mus musculus; RNA-Seq
For preparation of nuclear-enriched RNA, cells were grown to confluency on 15 cm plates (Falcon), treated with stress, washed in PBS, scraped in PBS, Pelleted by centrifugation at 3000 x g and resuspended in 200 uL of RLB buffer (10 mM Tris, pH 7.5, 140 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet P-40, 1mM DTT and 100U/ml RNase IN (Roche). Samples were incubated on ice for 5 min and then layered over 600 ul the RLB buffer containing 24% (wt/vol) sucrose.  Samples were centrifuged at 13000 x g for 10 min at 4°C. After centrifugation, supernatants (cytoplasmic extracts) were discarded and nuclear pellets were resuspended in 200 ul buffer w. sucrose, and RNA was extracted by the addition of 1 ml Trizol. RNA (total or nuclear) was then treated with DNase RQ (Promega) using 0.5-1 units DNase/μg RNA following the manufacturer’s instructions. RNA was recovered by phenol/chloroform/isoamyl alcohol (PCA) extraction and ethanol precipitation, following standard protocols. For sequencing, RNA was subjected to additional rounds of purification by Qiagen RNaEasy kit followed by PCA. RNA quality was assessed by NanoDrop 8000 (Thermo Scientific) and agarose gel electrophoresis. RNA quality was further established at the Yale Center for Genome Analysis (YCGA) by running an Agilent Bioanalyzer gel. Ribosomal RNA was then depleted using the Ribo-Zero Gold Kit (MRZG12324, Epicentre) and remaining RNA fragmented by incubation at 94°C. Strand-specific Libraries were prepared using Illumina reagents. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP for generating strand-specific sequencing libraries. The cDNA library was end-repaired and A-tailed, adapters were ligated and second-strand digestion was performed by Uricil-DNA-Glycosylase. Quality of indexed libraries was validated by quantification by qRT-PCR (KAPA Biosystems) and by insert size distribution determination (LabChip GX). Two samples were multiplexed per sequencing lane, and samples were sequenced using 75 bp paired-end sequencing on an Illumina HiSeq 2000 according to Illumina protocols. Signal intensities were converted to individual base calls during the run using the system’s Real-Time Analysis (RTA) software. Sample de-multiplexing and splitting of the paired-end reads were done using Illumina's CASAVA 1.8.2 software suite.