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Sample GSM2375977 Query DataSets for GSM2375977
Status Public on Mar 28, 2017
Title G2 heIF4A1 repA
Sample type SRA
Source name HEK293
Organism Homo sapiens
Characteristics expression vector: heIF4A1
clip antibody: Anti-FLAG® M2 Magnetic Beads (M8823 Sigma)
Extracted molecule total RNA
Extraction protocol The cells are rinsed with PBS and crosslinked with 0.15mJ/cm2 UV-C light. After crosslinking, the cells are pelleted by centrifugation, snap-frozen in liquid nitrogen and kept at -80˚C until use. Cells are lysed with 0.5mL of lysis buffer, mildly sonicated and immunoprecipitated with anti-FLAG beads for 1hr at ˚C. The beads are washed with lysis buffer and bound material is eluted with 3xFLAG peptide (250µg/mL). The eluate is then incubated with MyONEC1 beads to collect biotinylated target protein, after which the beads are washed with high-stringency buffers (0.1%SDS, 1M NaCl, 0.5% LiDS, 0.5M LiCl and 1% SDS, 0.5M LiCl) to aggressively remove non-specific interactors. 3’-linkers are then ligated with T4 RNA Ligase 1, excess adapters are washed away and 3’-tagged, cross-linked RNA is released with proteinase K digestion and column purification (Zymo DNA Clean & Concentrator).
Reverse transcription is carried out with SuperScript III and barcoded reverse-transcription primers. After reverse transcription, relevant samples are mixed and the cDNA is separated on a 6% 6M Urea PAA gel. Size fractionated cDNA is then processed essentially as described in the iCLIP protocol to generate sequencing libraries (König, J. et al. iCLIP--transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution. J. Vis. Exp. (2011). doi:10.3791/2638). uvCLAP reads contain two custom barcodes adjacent to the 5'- and 3'-adapters. The 5 nt 5'-barcodes are flanked by 5 random nt according to the pattern NNNTTTTTNN (N: random nucleotide, T: barcode nucletide) and specify the pulldown condition. The 5 nt semi-random 3'-barcodes DRYYR and DYRRY (D: not C, R: purine, Y: pyrimidine) are used to distinguish the two replicates of each experiment. The 5'-barcodes are located at the first 10 nucleotides of the first mate reads. The read pairs align forward/reverse, thus the reverse complement of the 3'-barcodes is located at the first 5 nucleotides of the second mate reads.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Description 5’-barcode: GAGGT, 3’-barcode: RYYR
processed data file: G2_heIF4A1_hg19_plus.bigWig
processed data file: G2_heIF4A1_hg19_minus.bigWig
processed data file: G2_heIF4A1_hg19.narrowPeak
Data processing Barcodes and random tags were extracted using custom fastqpl scripts (Horton P. fastapl – a utility for versatile and easy processing of fasta format data (and fastqpl for fastq files). F1000Posters 2011, 2:1197 (poster)).
Adapters were trimmed and the library was demultiplexed using Flexbar (v2.32).
Possible readthroughs into the barcoded regions were removed by clipping 10 and 5 nt from the 3’-ends of first and second mate reads.
Reads were mapped to the reference genomes (hg19, mm10, dm3) using bowtie2 (v2.2.0) with parameters --very-sensitive --end-to-end --no-mixed --no-discordant and --maxins=200.
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)).
Crosslinking events of M and L size fractions were combined for each biological replicate.
Peaks were called with JAMM (version 1.0.7rev1, parameters "-d y -t paired -b 50 -w 1 -m normal") on crosslinked nucleotides of the two replicates for each pulldown condition as foreground and the control condition as background.
Genome_build: hg19, dm3 or mm10 as indicated
Supplementary_files_format_and_content: bed files of crosslinking event alignments with random tag in name column and number of merged PCR-duplicates in score column; bigWig profiles of combined L and M fractions for both strands; JAMM peaks in narrowPeak format. BigWig and narrowPeak files are available in a tar archive on the series record. Bed files are available on the sample records.
Submission date Nov 04, 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 GPL11154
Series (2)
GSE85155 DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome [uvCLAP CLIP-seq]
GSE85164 DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome
BioSample SAMN05983755
SRA SRX2328712

Supplementary file Size Download File type/resource
GSM2375977_G2_heIF4A1_repA_hg19.bed.gz 793.1 Kb (ftp)(http) BED
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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