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| Status |
Public on Sep 18, 2017 |
| Title |
NET-seq DMSO Rep2 |
| Sample type |
SRA |
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| Source name |
DMSO
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MOLT4
library type: 3'end RNA fragment sequencing
treatment: DMSO
molecule subtype: nascent RNA
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| Growth protocol |
cells were cultured in RPMI-10%FCS
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| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions. External spike-in RNAs (ERCC ExFold RNA Spike-In kit, Ambion, 4456739) were added based on cell number. See manuscript for details.
For NET-seq, the library preparation is performed as described by Churchman and Weissman (2011, 2012) with modifications. For 30 RNA ligation, a pre-adenylated DNA linker with a mixed random hexameric barcode sequence at its 50 end is used. cDNA containing the 30 end sequences of a subset of mature and heavily sequenced snRNAs, snoRNAs, rRNAs, and mitochondrial tRNAs are specifically depleted using biotinylated DNA oligos, as described by Ingolia et al.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Reads are trimmed and aligned using STAR (v2.4.0) (Dobin et al., 2013)
For NET-seq data, only the position matching the 50 end of the sequencing read (after removal of the barcode), corresponding to the 30 end of the nascent RNA fragment, is recorded with a Python script using HTSeq package (Anders et al., 2015). Reverse transcription mispriming events are identified and removed when molecular barcode sequences match exactly to the genomic sequence adjacent to the aligned read. Reads that align to the same genomic position and contain identical barcodes are considered PCR duplication events and are removed. Splicing intermediates have 30 hydroxyls and will enter NET-seq libraries and contribute to the reads aligning to the exact single-nucleotide 30 ends of introns and 30 ends of exons.
Genome_build: hg19
Supplementary_files_format_and_content: Supplementary_files_format_and_content: BedGraph files (http://genome.ucsc.edu/goldenPath/help/bedgraph.html); Column1=chromosome; Column2=start (left most coordinate; 0 based; position included); Column3=end (right most coordinate; 0 based; position not included); Column4=Coverage (3'end nt for NET-seq and whole read for RNA-seq)
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| Submission date |
Mar 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
James Bradner |
| E-mail(s) |
bradner_computation@dfci.harvard.edu
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Medical Oncology
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| Lab |
Bradner Lab
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| Street address |
450 Brookline
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE79271 |
BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment [NET-seq] |
| GSE79290 |
BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment |
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| Relations |
| BioSample |
SAMN04558359 |
| SRA |
SRX1637483 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2090426_DMSO_rep2_netseq_neg.bedGraph.gz |
33.1 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM2090426_DMSO_rep2_netseq_pos.bedGraph.gz |
33.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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