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Sample GSM1422166

Query DataSets for GSM1422166

Status

Public on Mar 12, 2015

Title

Bl6_mESC_C2cells_2i_Ctrl_RNA-seq_rep2

Sample type

SRA

Source name

embryonic stem cell

Organism

Mus musculus

Characteristics

cell type: embryonic stem cell
treatment: control

Treatment protocol

To activate Cre recombinase and recombine out the floxed NELF-B allele, NELF-BFl/Fl, CreER+/- ESCs were treated with 100 nM 4OHT (Sigma) for 4 days (unless otherwise indicated). ESCs were then grown in 2i media without 4OHT for the completion of the experiment.

Growth protocol

All ESC culture was conducted at 37°C in 5% CO2. NELF-BFl/Fl, CreER+/- ESCs were maintained without feeders in 2i media (knockout DMEM (KO-DMEM), 15% knockout serum replacement (KOSR, Invitrogen), 1 mM NaPyruvate (Millipore), 1% NEAA, 1% BME, 1% Pen/Strep, 1% Glutamax, 1000 U/ml ESGRO, 1 µM MEK inhibitor (PD0325901, Stemgent), and 3 µM GSK3 inhibitor (CHIR99021, Stemgent)) and passaged every 2 days.

Extracted molecule

total RNA

Extraction 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).
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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

GRO-seq: GRO-seq reads were trimmed for quality and adapter sequence using cutadapt 1.2.1 , discarding pairs where either mate was trimmed shorter than 15 nt (-m 15 -q 10 --match-read-wildcards). Read pairs originating from rRNA were filtered by aligning against an index consisting of the 45S and 5S transcripts using Bowtie 0.12.8, allowing two mismatches (-m1 -v2 -X1000 --un --max). Unmapped pairs were subsequently aligned to the mm9 index, allowing two mismatches and retaining only uniquely aligned pairs (-m1 -v2 -X1000). BedGraph files were generated from the combined alignments of all technical and biological replicates, reduced to their strand-specific 5’-end mapping locations (N=59,099,712 reads: individual samples had mappable read depths of 33,123,684 for Library#1, and 25,976,028 for Library #2. Agreement between replicates was strong (R2=0.96, fig. S1A), thus the data was combined for further analysis.
RNA-seq: Read pairs were filtered, requiring a mean quality score greater than or equal to 20, for both mates, then aligned to the mm9 reference genome using TopHat 2.0.4, reporting up to 10 alignments per read pair (-g 10). Mean fragment sizes and standard deviations were determined using Picard Tools 1.86 CollectInsertSizeMetrics, based on a bowtie 0.12.8 alignment (-m1 -v2 -X10000) of a subset of one million reads to an index of RefSeq transcripts, and passed to TopHat using the --mate-inner-dist and --mate-std-dev parameters. A total of 113,817,860 and 108,809,117 read pairs were successfully aligned for the Control and 4OHT/NELF-B KO samples, respectively. UCSC Browser tracks displaying read coverage normalized per million mappable fragments were generated from the combined replicates per condition.
Genome_build: mm9
Supplementary_files_format_and_content: GRO-seq: BedGraph files were generated from the combined alignments of all technical and biological replicates, reduced to their strand-specific 5’-end mapping locations
Supplementary_files_format_and_content: RNA-seq: bigWig tracks displaying read coverage normalized per million mappable fragments were generated from the combined replicates per condition.

Submission date

Jun 27, 2014

Last update date

May 15, 2019

Contact name

Karen Adelman

E-mail(s)

[email protected]

Organization name

Harvard Medical School

Department

Biological Chemistry and Molecular Pharmacology