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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
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 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
Relations
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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