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Sample GSM1833635

Query DataSets for GSM1833635

Status

Public on Feb 01, 2017

Title

K1-ER 4sU-RNA-seq Rep 2

Sample type

SRA

Source name

K1-ER Clone C

Organism

Mus musculus

Characteristics

cell type: KLF1 null erythroblasts (K1)
transgene: KLF1-ER

Treatment protocol

Cells were induced through the addition of (Z)-4-Hydroxytamoxifen at a final concentration of 200 nM and newly transcribed RNA labeled with 500 μM 4sU for 1 h.

Growth protocol

Erythroid cell lines were passaged (1:5) every 2-3 days in DMEM supplemented with 10% FBS, 1% Glutamine & 1% Penicillin-Streptomycin in a standard tissue culture incubator at 37 °C with 5% CO2

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using TRIzol® Reagent (Life Technologies, #15596026) according to the manufacturer’s instructions with manually dissociated cells washed in 1 ml DPBS (Gibco™, #14190-136). Extracted RNA was subsequently treated with DNase I (New England Biolabs, #M0303L) to remove residual gDNA contamination. Isolation of 4sU-RNA was performed essentially as previously described (Rabani et al. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat Biotechnol 2011) with minor modifications. Purified RNA was biotinylated with EZ-Link Biotin-HPDP (Pierce; #21341) and isolated using µMacs Streptavidin Kit (Miltenyi; #130-074-101). 4sU-enriched RNA was then purified with RNeasy MinElute Spin Columns (Qiagen; #74204).
Purified 4sU-labelled RNA was used with the Ion Total RNA-Seq Kit v2 (Life Technologies; #4479789) to prepare libraries for sequencing on the Ion Proton™ System.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent Proton

Description

K1-ER_K1-NanER_DEGs.csv.gz
4sU-RNA

Data processing

Base calling was performed with Torrent Suite v4.0.1
[ChIP-seq]
TMAP (v3.0.1) with the following options: mapall, -v, -Y, -u, -o 2, stage1, map4 was used to map reads to the reference genome (mm9; ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm9/Mus_musculus_UCSC_mm9.tar.gz)
Fragments mapping to the same position on the same strand were taken to be PCR duplicates and were subsequently discarded, retaining only unique alignments with the highest MAPQ score.
ChIP-seq peaks were called with MACS (v1.4.2) using uniquely mapped ChIP and input sequence alignments.
Significant peaks identified by MACS were further filtered and ranked based on >30 fold-enrichment compared to input, and a false-discovery rate <0.1%. Peaks overlapping regions identified as RNA repeats by the Repeatmasker software were ignored, yielding lists of high-confidence peaks for each sample.
Each peak summit was subsequently annotated with the genomic context, overlapping peaks and central KLF1 binding motif.
[RNA-seq]
Tophat2 (v2.0.10) was used to perform spliced alignment to the reference transcriptome and genome (mm9; ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm9/Mus_musculus_UCSC_mm9.tar.gz) using default options with the following additional parameters: -N 4, --read-gap-length 4, --read-edit-dist 4, --read-realign-edit-dist 0, -a 5, -i 50, -g 6, --coverage-search, --microexon-search, --library-type fr-firststrand.
Unaligned sequences were subsequently remapped using TMAP (v3.0.1) with the following options: mapall, -v, -Y, -u, -o 2, stage1, map4.
Successful alignments were merged back into the Tophat2 SAM file.
Alignments of less than 30 nt were discarded.
Fragments Per Kilobase of transcript per Million mapped reads (FPKM) was calculated and differential expression testing between replicate groups was performed with Cuffdiff2 using default parameters. A mean FPKM cutoff of 1.0 was required in at least one sample and only differentially expressed genes (DEGs) with >2 fold-change were reported.
Genome_build: mm9
Supplementary_files_format_and_content: Nan-KLF1_Annotated_Peaks.csv.gz is a tab-delimited annotated MACS peaks file
Supplementary_files_format_and_content: KLF1_Annotated_Peaks.csv.gz is a tab-delimited annotated MACS peaks file
Supplementary_files_format_and_content: K1-ER_K1-NanER_DEGs.csv.gz reports tab-delimited FPKM values and differential gene expression analysis
Supplementary_files_format_and_content: Nan_FL_DEGs.csv.gz reports tab-delimited FPKM values and differential gene expression analysis

Submission date

Jul 27, 2015

Last update date

May 15, 2019

Contact name

Kevin Robert Gillinder

Organization name

Monash University

Department

Australian Centre for Blood Diseases

Street address

99 Commercial Road