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| Status |
Public on Feb 04, 2022 |
| Title |
HEK293T par-clip from HDLBP_2 |
| Sample type |
SRA |
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| Source name |
HEK293 Flp-In T-REx embryonic kidney cell line
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| Organism |
Homo sapiens |
| Characteristics |
genotype/variation: FLAG/HA tagged HDLBP
cell line: HEK293T
replicate: replicate 1
molecule subtype: HDLBP-bound RNA fragment
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| Treatment protocol |
FLAG/HA-HDLBP-HEK293 Flp-In T-REx cells were incubated with 100 µM 4sU for 16 h.
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| Growth protocol |
FLAG/HA-HDLBP-HEK293 Flp-In T-REx cells were cultured in standard Dulbeco´s modified Eagle´s medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% 2 mM L-glutamine (Thermo Fisher Scientific).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were UV crosslinked (365 nm, 0.15 J/cm²), harvested, snap frozen and stored at -80°C. Cell pellets were incubated with 3 cell pellet volumes of lysis buffer (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 % IGEPAL CA-630, 0,1 % SDS, 0,5% sodium deoxycholate, complete EDTA-free protease inhibitor cocktail [Roche]) for 30 min on ice. Cell lysates were cleared (13.000 rpm, 15 min, 4°C), filtered and RNase T1 was added at final concentration of 1 unit/µl for 10 min at 22°C. 20 µl beads (Dynabeads Protein G, Thermo Fisher Scientific) per 1 ml lysis buffer were washed twice with lysis buffer and coated for 1 h with 0,25 µg/µl FLAG antibody (Sigma-Aldrich, F1804). Afterwards beads were washed with lysis buffer twice and added to the sample. After 2 h incubation at 4°C on a rotating wheel the beads were washed with IP wash buffer (50 mM HEPES-KOH [pH 7.5], 300 mM KCl, 0,05 % IGEPAL CA-630, 0,5 mM DTT, complete EDTA-free protease inhibitor cocktail) 3 times and a second RNase T1 treatment was carried out. For replicate 1 a final concentration of 1 unit/µl and for replicate 2 a final concentration of 15 unit/µl was used for 10 min at 22°C. Subsequently, beads were washed with high-salt was buffer (50 mM Tris-HCl [pH 7.4], 1 M NaCl, 1 % IGEPAL CA-630, 0,1 % SDS, 0,5 % sodium deoxycholate, 1 mM EDTA, complete EDTA-free protease inhibitor cocktail [Roche]) 3 times and 2 times with polynucleotide kinase buffer (50 mM Tris-HCl [pH 7.5], 50 mM NaCl,10mM MgCl2, 5 mM DTT). Samples were radiolabeled using T4 polynucleotide kinase (NEB) and resolved on a Novex 4-12% Bis Tris gel (Thermo Fischer Scientific). The protein-RNA complexes were transferred to a nitrocellulose membrane (Whatman), exposed to a phosphorimager screen for 30 min and excised at approximately 160 kDa. After proteinase K (Roche, 40 min at 50°C) digest the RNA was extracted by phenol-chloroform treatment and precipitation.
small RNA cloning and library generation (Hafner et al.,2012, PMID:22926237) using 3′ adaptor 4N-RA3, 5′ adaptor OR5-4N, RT primer RTP and PCR primers RP1 (forward primer) and RPI1-12 (reverse primer, containing barcodes, for sequences see Illumina Truseq small RNA oligonucleotides)
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Library strategy: PAR-CLIP
Basecalls were converted to fastq files using Bcl2Fastq (2.16.0.10). Reads were demultiplexed, stripped of the 3’adapter sequence by Flexbar (v2.5) and collapsed to remove PCR duplicates. This was followed by trimming of four nucleotides from both 5’ and 3’ end of the read using FASTX Toolkit v0.0.14. Next, the reads were aligned to the human genome (hg19 build) using BWA v0.7.15-r1140 by using the previously published computational PAR-CLIP pipeline (v0.97a) (Lebedeva et al. 2011; Jens 2016). https://github.com/marvin-jens/clip_analysis.
To obtain gene level binding information, we summed T-C transitions in reproducible clusters for each gene. We thus obtained the total number of crosslink positions within whole mRNAs, or within the CDS, 5’UTR or 3’UTR.
To assess the relative distribution of TC-transitions within the transcriptome and obtain accurate mapping to transcripts originating from repetitive genomic loci, reads were sequentially mapped to reference transcripts by Bowtie2 (v2.3.2) in the following order by retaining the unmapped reads from the previous to the next mapping step. We started with human pre-rRNA (GenBank U13369.1), followed by rRNA (GenBank NR_023363.1, NR_003285.2, NR_003287.2, NR_003286.2), snRNA, snoRNA, other ncRNA (all from Ensembl, including RN7SL), tRNA (GtRNADb), mtDNA (GenBank AF347015.1) and finally the human genome (hg19, iGenomes). The last genome-mapping step was performed by the STAR aligner (v2.2.1) where only uniquely mapped reads (MAPQ=255) were retained. Except for the tRNA mapping (see below for details), we retained reads that mapped with a Bowtie2 MAPQ=20 or more and T-C transitions were extracted using row_mpile_coverage_plus_TC.pl script (Schueler et al. 2014).
For transcriptome level analysis, remaining reads after mtDNA mapping were aligned to the transcriptome sequence (GTF annotation file Gencode v19) by the STAR aligner (v2.2.1) by retaining only the reads that uniquely mapped to the hg19 genome (parameters --outFilterMultimapNmax 1 --outFilterMismatchNmax 5 --outFilterMatchNmin 15 --alignSJoverhangMin 5 --seedSearchStartLmax 20 --outSJfilterOverhangMin 30 8 8 8 --quantMode TranscriptomeSAM). T-C transitions with transcriptome coordinates were obtained from the BAM file using the row_mpile_coverage_plus_TC.pl script (Schueler et al. 2014) and written into a bed file.
3’ adaptor 4N-RA3, rApp-NNNNTGGAATTCTCGGGTGCCAAGG-InvdT; 5’ adaptor OR5-4N, rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrCrNrNrNrN
Genome_build: hg19
Supplementary_files_format_and_content: processed_data_parclip_pergene.txt: tab delimited matrix of T-C transitions, unique read counts and cluster counts in the coding sequence (CDS), 3' untranslated regions (3'UTR), 5' untranslated regions (5'UTR) and whole mRNA (transcript) (mean of 2 replicates) for all detected genes.
Supplementary_files_format_and_content: processed_data_parclip_per_transcript.txt: tab delimited matrix of T-C transitions in transcript coordinates, number of T-C transitions and total read coverage for every position in both replicates, sequence of the crosslink-centred 7-mer, normalized number of T-C transitions, lengths of mRNA, 5'UTR, CDS and 3'UTR (l_tr, l_utr5,l_cds,l_utr3), transcript coordinates of CDS start and stop, as well as corresponding gene id and transcript expression level (TPM_transcript).
Supplementary_files_format_and_content: processed_data_parclip_trna.txt: tab delimited matrix of T-C transitions in tRNA coordinates (tc_start, tc_stop), number of T-C transitions and total read coverage for every position in both replicates (TCnumber_hdlbp2 TCnumber_hdlbp3, allReadsPerPos_hdlbp2 allReadsPerPos_hdlbp3), sequence of the crosslink-centred 7-mer (seq_tc_middle_t), normalized number of T-C transitions, mean fraction of T-C transitions over total coverage, standard deviation of fraction of T-C transitions over total coverage, T-C transition signal in whole tRNA normalized to the total tRNA abundance.
Supplementary_files_format_and_content: processed_data_parclip_overlapping_clusters.txt: tab delimited matrix of HDLBP binding clusters for both replicates (1 and 2) in chromosome coordinates, including cluster start, stop, cluster id, number of T-C transitions in cluster (tc_rep), strand, number of unique reads in cluster, chromosome coordinate of cluster centered region, number of overlapping nucleotides between cluster replicates, corresponding gene and region (CDS, 3'UTR, 5'UTR, start codon, stop codon).
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| Submission date |
Apr 07, 2020 |
| Last update date |
Feb 04, 2022 |
| Contact name |
Markus Landthaler |
| E-mail(s) |
markus.landthaler@mdc-berlin.de
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| Organization name |
Max Delbrueck Center
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| Department |
Berlin Institute for Medical Systems Biology
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| Street address |
Hannoversche Str. 28
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| City |
Berlin |
| ZIP/Postal code |
10115 |
| Country |
Germany |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE148259 |
Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (HDLBP PAR-CLIP) |
| GSE148262 |
HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins |
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| Relations |
| BioSample |
SAMN14554483 |
| SRA |
SRX8077289 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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