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| Status |
Public on Mar 28, 2017 |
| Title |
XL2 hnRNPC-mAb-HEK rep A |
| Sample type |
SRA |
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| Source name |
HEK293
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
clip antibody: hnRNPC(4F4) (Santa cruz, cat#: sc-32308, lot: D2914)
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| 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.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
3'-tag: TTAGCC, 3'-RY-space tag: RR
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| 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).
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.
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| Submission date |
Feb 10, 2017 |
| 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 |
GPL18573 |
| Series (2) |
| GSE85164 |
DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome |
| GSE94781 |
DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome [hnRNPC FLASH CLIP-seq] |
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| Relations |
| BioSample |
SAMN06320314 |
| SRA |
SRX2550929 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2482824_XL2-hnRNPC-mAb-HEK-repA.bed.gz |
44.0 Mb |
(ftp)(http) |
BED |
| GSM2482824_XL2-hnRNPC-mAb-HEK-repA_minus.bigWig |
12.5 Mb |
(ftp)(http) |
BIGWIG |
| GSM2482824_XL2-hnRNPC-mAb-HEK-repA_plus.bigWig |
13.2 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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