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Sample GSM2387774 Query DataSets for GSM2387774
Status Public on Mar 28, 2017
Title XL8 IgG-Rb ESC repA
Sample type SRA
Source name embryonic stem cells
Organism Mus musculus
Characteristics cell type: embryonic stem cells
cell line: WT26
strain: C57BL/6x129sV
clip antibody: IgG-Rb (Santa Cruz, sc-2027m lot H2814)
Treatment protocol The feeder-independent mouse male ES cell line WT26 (a kind gift from the lab of T. Jenuwein), was grown on gelatin-coated dishes in ES cell culture media KnockOut-DMEM supplemented with 1% L-glutamine, 1% penicillin/streptavidin, 1% Non- essential amino acids, 1% Sodium Pyruvate, 1% 2-mercaptoethanol. All ESC media contained 15% FBS and 1000 U/ml (for feeder free) or 2000U/ml (for feeder-dependent) of leukemia inhibitory factor (LIF).
Extracted molecule total RNA
Extraction protocol Cells are crosslinked with 0.15mJ/cm2 UV-C irradiation, lysed with 1xNLB and homogenized by waterbath-sonication. Target protein of interest is pulled-down with paramagnetic beads pre-coupled to antibodies against the protein of interest.
After a brief RNAseI-digestion, RNA 3'-ends are healed with T4 PNK. Custom-made, barcoded adapters are ligated using T4 RNA Ligase 1 or T4 RNA Ligase 2KQ (if pre-adenylated adapters are used) for 1 hr at 25˚C. Custom FLASH adapters contained two barcodes and random nucleotides adjacent to the 3'-adapters according to the pattern NNBBNTTTTTTNN (N: random tag nucleotide, T: tag nucleotide, B: RY-space tag nucleotide). Random tags are used to merge PCR-duplicates, regular tags are used to specify the pulldown condition, and semi-random RY-space tags are used to distinguish the biological replicates (RR: replicate A, YY: replicate B, R: purine, Y: pyrimidine). Excess adapters are washed away, negative controls (IgG) are mixed with experimental controls and RNA is isolated with Proteinase K treatment and column purification. Isolated RNA is reverse-transcribed and RNaseH-treated. cDNA is column-purified and circularized with CircLigase for 2-16hrs. Circularized cDNA is directly PCR amplified, quantified with Qubit / Bioanalyzer and sequenced on Illumina NextSeq 500 in paired-end mode.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description 3'-tag: TTAGCC, 3'-RY-space tag: RR
Data processing Adapters were trimmed using Flexbar (v2.5).
Libraries were demultiplexed using bctools (, v0.2.0) and Flexbar (v2.5).
Possible readthroughs into the barcoded regions were removed by clipping 13 nt from the 3'-ends of first mate reads.
Reads were mapped to reference genome hg19 using bowtie2 (v2.2.6) with parameters --very-sensitive --end-to-end --no-mixed --no-discordant --maxins 500.
Alignments of uniquely mapped reads were extracted and used to determine crosslinking events as previously described (Ilik, I. A. et al. Tandem Stem-Loops in roX RNAs Act Together to Mediate X Chromosome Dosage Compensation in Drosophila. Mol. Cell 51, 156–173 (2013)) with bctools (, v0.2.0).
Peaks for DHX9-Rb were called with PEAKachu (, v0.0.1alpha2) using parameters --pairwise_replicates, --max_insert_size 100, -m 0, -n manual, and --size_factors 1 1 0.75 0.75, with the IgG control as experimental background.
Genome_build: mm10
Supplementary_files_format_and_content: bed files containing coordinates of crosslinking event alignments with id of a representative read in name column and number of merged PCR-duplicates in score column. bigWig profiles of crosslinking event alignments for each strand. PEAKachu peaks in gff format.
Submission date Nov 10, 2016
Last update date May 15, 2019
Contact name Asifa Akhtar
Organization name Max Planck Institute of Immunobiology and Epigenetics
Department Chromatin Regulation
Lab Akhtar Lab
Street address Stuebeweg 51
City Freiburg
ZIP/Postal code 79108
Country Germany
Platform ID GPL19057
Series (2)
GSE85164 DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome
GSE89751 DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome [XL8 DHX9 FLASH CLIP-seq]
BioSample SAMN06009440
SRA SRX2341512

Supplementary file Size Download File type/resource
GSM2387774_XL8_IgG-Rb_ESC_repA.bed.gz 84.4 Kb (ftp)(http) BED
GSM2387774_XL8_IgG-Rb_ESC_repA_minus.bigWig 56.7 Kb (ftp)(http) BIGWIG
GSM2387774_XL8_IgG-Rb_ESC_repA_plus.bigWig 59.9 Kb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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