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Status |
Public on Mar 28, 2017 |
Title |
G2 hnRNP-K repB |
Sample type |
SRA |
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Source name |
HEK293
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Organism |
Homo sapiens |
Characteristics |
expression vector: hnRNP-K clip antibody: Anti-FLAG® M2 Magnetic Beads (M8823 Sigma)
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
5’-barcode: TCACG, 3’-barcode: YRRY processed data file: G2_hnRNP-K_hg19_plus.bigWig processed data file: G2_hnRNP-K_hg19_minus.bigWig processed data file: G2_hnRNP-K_hg19.narrowPeak
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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.
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Submission date |
Nov 04, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Asifa Akhtar |
E-mail(s) |
akhtarlab_data@ie-freiburg.mpg.de
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Organization name |
Max Planck Institute of Immunobiology and Epigenetics
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Department |
Chromatin Regulation
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Lab |
Akhtar Lab
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Street address |
Stuebeweg 51
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City |
Freiburg |
ZIP/Postal code |
79108 |
Country |
Germany |
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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 |
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Relations |
BioSample |
SAMN05983758 |
SRA |
SRX2328715 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2375980_G2_hnRNP-K_repB_hg19.bed.gz |
19.8 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
Processed data are available on Series record |
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