title: GSM1551911: Adelman_Bl6_mESC_C2cells_2i_4OHT_startRNA-seq_rep1; Mus musculus; RNA-Seq
library_construction_protocol: GRO-seq: GRO-seq experiments were performed according to {Core, 2008 #1330} in the C2 ESC line grown in 2i media, with the following modifications: Nuclei were harvested by swelling trypsinized cells for 10 minutes in 50 mL cold lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM CaCl2, 2 mM MgCl2, 0.5% NP40, 5 mM DTT, 300 mM sucrose, 1 mM PMSF, 2 U/ml Superase IN, 5 tablets Roche protease inhibitors), then gently dounce homogenized, centrifuged and washed once with 50 mL lysis buffer.  Nuclei were resuspended in freezing buffer as in {Core, 2008 #1330} at the concentration of 5x106 nuclei per 100 uL, and stored at -80°C until use. To avoid a bias against representation of C-rich regions in the data set, the final concentration of CTP (Roche) was increased in the run-on ten-fold above that used in {Core, 2008 #1330} to 10 uM. To permit a quick, reproducible stop to the run-on reaction, the reaction was terminated after 5 minutes by the addition of Trizol (Invitrogen). RNA-seq: Two distinct conditional NELF-B KO ESC clones were grown +/- 4OHT in 2i media. On day 5 after initiating recombination, total RNA was prepared using Trizol (Invitrogen) and manufacturer recommended methods. DNA contamination was removed using the RNeasy Mini Kit (Qiagen) per manufacturer’s instructions, and RNA quality was assessed using a Bioanalyser Nano ChIP (Agilent). startRNA-seq: Total RNA was extracted from nuclei using Trizol reagent (Invitrogen) GRO-seq: Following base hydrolysis, primary Br-U immunoprecipitation and end repair, the libraries were made using the Illumina small RNA TruSeq kit except the 3’ adapter was ligated using T4 RNA Ligase II, truncated KQ enzyme (NEB).  A secondary BrU IP was performed before ligation of the 5’ adapter (Illumina) followed by a tertiary BrU IP to produce triple-selected run-on RNA.  RNA was reverse transcribed and amplified with 15 cycles of PCR (Illumina).  Libraries were PAGE purified, selecting for products larger than 140 bp. RNA-seq: Ribosomal RNA was removed prior to library construction by hybridizing to ribo-depletion beads that contain biotinylated capture probes (Ribo-Zero, Epicentre). RNA was then fragmented and libraries were prepared according to the TruSeq Stranded Total RNA Gold Kit (Illumina) using random hexamer priming. Illumina Spike-ins were used for normalization. startRNA-seq: Start-RNA libraries were prepared as described in (Nechaev et al., Science 2010) except reads were size selected in the range 20-80 nt to exclude full length snRNA species.