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Sample GSM2480886

Query DataSets for GSM2480886

Status

Public on May 31, 2018

Title

RNA_CTR_2

Sample type

SRA

Source name

RNA_CTR

Organism

Mus musculus

Characteristics

cell type: E14 embryonic stem cell (ESC)
genotype/variation: GFP

Growth protocol

E14 mouse embryonic stem cells were grown under standard serum/LIF conditions (DMEM 4,500 mg/l glucose, 4 mM L-glutamine, 110 mg/l sodium pyruvate, 15% fetal bovine serum, 1 U/ml penicillin, 1 mg/ml streptomycin, 0.1 mM nonessential amino acids, 50 mM b-mercaptoethanol, and 103 U/ml LIF).

Extracted molecule

total RNA

Extraction protocol

(RNA-Seq)Prior to RNA isolation cell lines carrying different Tet3 constructs were enriched for cells with TET3 expression by flow sorting for GFP fluorescence using a BD Influx High-speed Cell Sorter. RNA was isolated using Qiagen RNeasy Micro columns and treated with DNaseI (Ambion).
(ChIP-Seq)Chromatin immunoprecipitation was performed as described in (Schmidt et al. 2009). ES cell lines expressing different TET3 isoforms were cross-linked and harvested. 10 ug of a TET3 antibody developed with Millipore (ABE290) was used per precipitation, and cross-linked, sonicated nuclear material was used as input control.
(ATAC-Seq)ATAC-seq was performed according to the original protocol (Buenrostro et al., 2015) with the following modifications. 10,000 cells were treated with 0.5μl Tn5 transposase in a total volume of 20 μl for 30 minutes at 37 degrees.
(RNA-Seq)Indexed RNA-Seq libraries were constructed from 500 ng total RNA using theTruSeq Stranded Total RNA Library Prep Kit for Illumina with Ribo-Zero Gold.
(ChIP-Seq)Sequencing libraries from precipitated DNA were prepared using the Diagenode MicroPlex Library Preparation Kit.
(ATAC-Seq)Sequencing libraries were prepared according to the original protocol (Buenrostro et al., 2015). A total of 15 PCR cycles and two rounds of Ampure Bead clean-up were performed.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

total RNA rRNA depleted
RPM_log2_RNA-Seq.txt

Data processing

Libraries were sequenced on the Illumina HiSeq 2500 platform.
(RNA-Seq) Reads were trimmed using Trim Galore v0.4.2 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) using default parameters to remove the standard Illumina adapter sequence. They were mapped to the mouse GRCm38 genome assembly using Tophat 2.0.12 (options -g2) guided by the gene models from the Ensembl v70 release. BAM files were imported to Seqmonk v36.0. (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) .
(ATAC-Seq) Reads were trimmed using Trim Galore v0.4.2 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) using default parameters to remove the standard Illumina adapter sequence. They were mapped to the mouse GRCm38 genome assembly using Bowtie 2 v2.2.5 (default parameters) . BAM files were imported to Seqmonk v36.0. (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) .
(ChIP-Seq) Reads were trimmed using Trim Galore v0.4.2 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) using default parameters to remove the standard Illumina adapter sequence. They were mapped to the mouse GRCm38 genome assembly using Bowtie 2 v2.2.5 (default parameters) . BAM files were imported to Seqmonk v36.0. (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) .
Genome_build: GRCm38
Supplementary_files_format_and_content: (RNA-Seq) Quantitated RNA-Seq data file is tab delimited and contains the following columns: (1) gene name (Ensembl) ; (2) Chromosome; (3) Start; (4) End; (5) Strand; (6) ID (Ensembl) (7) Description (Ensembl) ; (8-16) log2 RPM value.
Supplementary_files_format_and_content: (ChIP-Seq) Quantitated ChIP-Seq data file is tab delimited and contains the following columns: (1) Chromosome; (2) Start; (3) End; (4-12) log2 RPM value per 500 bp tile.
Supplementary_files_format_and_content: (ATAC-Seq) Quantitated ATAC-Seq data file is tab delimited and contains the following columns: (1) Chromosome; (2) Start; (3) End; (4-9) log2 RPM value per 500 bp tile.

Submission date

Feb 08, 2017

Last update date

May 15, 2019

Contact name

Christel Krueger

E-mail(s)

christel.krueger@babraham.ac.uk

Organization name

Babraham Institute

Street address

Babraham Research Campus