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Sample GSM2866862

Query DataSets for GSM2866862

Status

Public on Mar 09, 2018

Title

Adelman_Dmel_S2_Start-seq_TSScall_rep2

Sample type

SRA

Source name

Adelman_Dmel_S2_Start-seq_TSScall

Organism

Drosophila melanogaster

Characteristics

cell line: S2
treatment: control
molecule subtype: nascent RNA

Treatment protocol

4sU experiments: Two independent biological replicates were generated for Control and 4OHT treated C2 embryonic stem cells to ablate NELF-KO where RNA was metabolically labeled for 10minutes using 500 μM 4-thiouridine. 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 5 days. ESCs were then grown in 2i media without 4OHT for the completion of the experiment. Start-seq experiments: Start-RNA libraries were prepared as described in (Nechaev et al., Science 2010 and Henriques et al. 2013). ChIP-seq experiments: ChIP-seq libraries were prepared as described in (Muse et al., Nat Genetics 2007 and Henriques et al. 2013).

Growth protocol

Drosophila S2 cells from the DGRC were grown in M3 media supplemented with bactopeptone, yeast extract and 10% FBS. For all experiments, cells were harvested at a consistent cell density of 4-6x10^6 cells/ml. mESC 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

4sU experiments: metabolically labeled RNA was extracted with Trizol. Equal amounts of total RNA were biotinylated and pulled down with streptavidin beads. 4-thiouridine labeled RNA was eluted with DTT, ethanol precipitated and resuspended in RNAse-free H2O. Start-seq experiments: nascent RNA was isolated from nuclei. ChIP-seq experiments: Sonicated genomic DNA was extracted after IP with respective antibody, Phenol-Chloform extracted and ethanol precipitated.
4sU experiments: TruSeq Stranded Total RNA, with Ribozero Gold and ERCC spikes. Start-seq experiments: TruSeq Small RNA, with homemade spike-in RNAs. ChIP-seq experiments: NextFlex Rapid DNA-seq on IPed or digested genomic DNA

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina MiSeq

Description

short capped RNA isolated from drosophila S2 nuclei
processed data file:
Adelman_Dmel_S2_Start-seq_TSScall_rep1to4_GSM463298_5prRNA_forward.bedGraph.gz
Adelman_Dmel_S2_Start-seq_TSScall_rep1to4_GSM463298_5prRNA_reverse.bedGraph.gz

Data processing

Base calling and generation of FASTQ files performed using standard CASAVA pipeline for HiSeq runs, bcl2fastq2 for NextSeq
4sU read pairs containing one or more members with mean quality score <20 filtered; start-seq trimmed for adapter using cutadapt 1.9.1, pairs containing reads trimmed shorter than 20 nt filtered
4sU mapped to reference using tophat 2.0.4, in a strand-specific manner, with bowtie1 as the underlying aligner, also to index of ERCC spikes using bowtie 0.12.8; start-seq mapped to index composed of annotated rRNA/tRNA and spike-in sequences, successfully aligned pairs filtered, remaining mapped to reference using bowtie 0.12.8 retaining uniquely mappable pairs only, allowing 2 mismatches; ChIP-seq mapped using bowtie 0.12.8 retaining uniquely mapped pairs only, allowing 2 mismatches
4sU strand-specific coverage tracks were generated using genomeCoverageBed, normalized with custom scripts using factors determined by DESeq 1.12.2 based on ERCC spike counts and combined with unionBedGraphs and custom scripts (mean count); start-seq strand-specific bedGraphs generated using custom scripts based on 5' mapping location of end 1 reads only, with all replicates combined; ChIP-seq bedGraphs were generated by removing duplicate fragments, filtering fragments of length <50 and >400, and determining counts of fragment centers in 25 nt bins tiling the genome, using custom scripts
Genome_build: dm3 (ChIP-seq, start-seq), mm9 (4sU)
Supplementary_files_format_and_content: 4sU: bigWig containing mean normalized coverage of all replicates; start-seq: bedGraph containing combined count of end 1 5' mapping locations for all replicates; ChIP-seq: bedGraph containing combined count of fragment centers for all replicates

Submission date

Nov 28, 2017

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