title: GSM4058539: CD14+.GRO.control.rep1; Homo sapiens; OTHER
library_construction_protocol: Cells were treated with medium containing 1% FBS. Cells washed twice with cold PBS and incubated on ice with 10mL of swelling buffer [10 mM Tris-HCl, 2 mM MgCl2, 3 mM CaCl2 and 2 U/ml SUPERase Inhibitor (Thermofisher, Carlsbad, CA, U.S.A.)] for 5 mins. Cells were scraped and centrifuged at 400xg for 10 min and resuspended in 50μL of swelling buffer supplemented with 10% glycerol. Next, 500μL of swelling buffer containing 10% glycerol and 1% Igepal was added drop by drop while gently vortexing. Nuclei was washed twice with lysis buffer (10 ml of swelling buffer containing 0.5% Igepal and 10% glycerol), and once with 1 ml of freezing buffer (50 mM Tris-HCl pH 8.3, 40% glycerol, 5 mM MgCl2 and 0.1 mM EDTA). Finally, nuclei were counted, centrifuged at 900xg for 6min to be further resuspended in to a concentration of 5 million nuclei per 100μL of freezing buffer, snap-frozen in liquid nitrogen and stored at -80°C until run-on reaction. The run-on reaction  was performed as described in [M. Bouvy-Liivrand et al. Analysis of primary microRNA loci from nascent transcriptomes reveals regulatory domains governed by chromatin architecture, Nucleic Acids Res. 23 (2017) 862–876.]. Library preparation were performed as described in [M. Bouvy-Liivrand et al. Analysis of primary microRNA loci from nascent transcriptomes reveals regulatory domains governed by chromatin architecture, Nucleic Acids Res. 23 (2017) 862–876.].  Libraries were amplified using 13-15 cycles, size-selected (180-350 bp) from 10 % TBE gels (Life Technologies) and sequenced (single end 50 bases) using Illumina HiSeq 2000 at EMBL GeneCore (Heidelberg, Germany). GRO-seq: Nuclei were extracted from 5-10 million cells and nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and BrUTP. RNA was extracted with Trizol-LS. RNA-seq, miRNA-seq: One hour after collection of GRO-Seq samples (reflecting the lag in nascent transcription and mature RNAs), cells were treated for 10 min with cycloheximide (0,1mg/mL), washed with PBS and scraped into lysis buffer [1x Mammalian Polysome Buffer (Epicentre, Madison, Wisconsin), 1% Triton X-100, 1 mM DTT, 250 U/ml SUPERase Inhibitor, 7.1 U/ml Turbo DNase (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.) and 0.1 mg/ml Cycloheximide] on ice. To confim the complete lysis of the cells, they were drawn up and expelled 4 times through a sterile 22-25 gauge needle. The cleared lysate was further treated with 10% SDS, snap-frozen in liquid nitrogen and stored at -80°C. For RNA-Seq, larger mRNAs (>200 nt) was purified with Zymo RNA Clean and Conc kit (Zymo Research, Irvine, California, U.S.A.) and rRNAs were eliminated using the Ribo-Zero Gold rRNA Removal Kit (Illumina, San Diego, CA, U.S.A.). For miRNA-Seq, small RNA-fraction (17-200 bases) was obtained by adding ethanol to the flow through from Zymo RNA Clean and Conc kit and passing the RNA through a new column. GRO-seq, RNA-seq: The RNA was fragmented (RNA fragmentation reagent, Thermofisher), dephosphorylated with PNK and purified using RNA Clean & Concentrator kit (Zymo). For GRO-Seq only immunopurification step was performed, where BrUTP-labeled run-on RNA was bound with anti-BrdUTP-coated agarose beads and precipitated overnight. FOr both GRO-Seq and RNA-Seq libraries, the RNA was polyadenylated, followed by cDNA synthesis using complementary poly(T)-primers containing Illumina adapter sequences. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified using ChIP DNA Clean & Concentrator kit. The recovered cDNA was RNaseH treated and circularized (CircLigase) and amplified for 11-16 cycles. The final product was ran on 10% TBE gel, gel purified (180-350 bp) and cleaned-up using ChIP DNA clean & Concentrator Kit.
attributes.tag: GEO Accession
attributes.value: GSM4058539