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Sample GSM2363522 Query DataSets for GSM2363522
Status Public on Mar 28, 2017
Title XL1 IgG-Rb rep A
Sample type SRA
 
Source name HEK293_XL1 IgG-Rb
Organism Homo sapiens
Characteristics cell line: HEK293
clip antibody: IgG-Rb
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 RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description 3'-tag: GTGGAA, 3'-RY-space tag: RR
Data processing Adapters were trimmed using Flexbar (v2.5).
Libraries were demultiplexed using bctools (https://github.com/dmaticzka/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 (https://github.com/dmaticzka/bctools, v0.2.0).
Peaks for DHX9-Rb and DHX9-mAb were with PEAKachu (https://github.com/tbischler/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: hg19
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 Oct 28, 2016
Last update date May 15, 2019
Contact name Asifa Akhtar
E-mail(s) akhtarlab_data@ie-freiburg.mpg.de
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 GPL18573
Series (2)
GSE85164 DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome
GSE89276 DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome [XL1 DHX9 FLASH CLIP-seq]
Relations
BioSample SAMN05950938
SRA SRX2279761

Supplementary file Size Download File type/resource
GSM2363522_XL1_IgG_Rb_repA.bed.gz 446.8 Kb (ftp)(http) BED
GSM2363522_XL1_IgG_Rb_repA_minus.bigWig 195.3 Kb (ftp)(http) BIGWIG
GSM2363522_XL1_IgG_Rb_repA_plus.bigWig 209.1 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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