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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 09, 2023 |
| Title |
ZFR-SMI-1 |
| Sample type |
SRA |
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| Source name |
HEK-293 Flp-In TREx cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK-293 Flp-In TREx cells
genotype: FLAG-ZFR stably integrated in FRT site
sample type: size-matched input
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| Treatment protocol |
Polyclonal cells were treated with doxycyline hyclate (1 μg/mL final concentration, ThermoFisher Scientific) for 48-72 hours for induction of transgene expression.
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| Growth protocol |
Flp-In T-REx-293 cells (ThermoFisher Scientific) were maintained at 37°C in ambient oxygen and 5% CO2 in DMEM (Gibco #11965‐092) supplemented with 10% fetal bovine serum (Gibco) and a 1% penicillin, streptomycin, and L‐glutamine mixture (Gibco).
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| Extracted molecule |
other |
| Extraction protocol |
Samples were prepared according to the eCLIP protocol (Van Nostrand et al. 2016 Nat Methods.) For each sample, 2x10^7 cells were UV crosslinked at 400mJ/cm2 constant energy. Cells were lysed on ice for 15 min in 1 mL lysis buffer containing 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, Halt protease inhibitor cocktail (ThermoFisher Scientific). Cells were passed through 27-gauge needles several times before sonication (Biorupter, Diagenode) for 3 min at low setting with 30 second on/30 second off duty cycle at 4°C.
eCLIP and size-matched input libraries were constructed according to the eCLIP protocol (Van Nostrand et al. 2016 Nat Methods.)
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
library strategy: eCLIP-seq
RNA-Seq data from eCLIP IP and input samples were analyzed using the ENCODE eCLIP-seq Processing Pipeline (Van Nostrand et al., 2018).
Raw fastq files were trimmed with Cutadapt as described (Martin, 2011), followed by removal of sequences derived from repetitive elements with STAR aligner, using human RepBase sequences (Bao et al., 2015; Dobin et al., 2013).
Remaining reads were mapped to the hg19 human genome release with STAR aligner.
ZFR eCLIP peaks were called with Clipper software and normalized vs the size-matched input control as described (Van Nostrand et al., 2016).
Round 1 trimming parameters: cutadapt -f fastq --match-read-wildcards --times 1 -e 0.1 -O 1 --quality-cutoff 6 -m 18 -a NNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -g CTTCCGATCTACAAGTT -g CTTCCGATCTTGGTCCT -A sequences corresponding to 15 nt tiling of X1A and X1B adapters
Round 2 trimming parameters:
cutadapt -f fastq
--match-read-wildcards --times 1 -e 0.1 -O 5 --quality-cutoff 6 -m 18 -A sequences corresponding to 15 nt tiling of X1A and X1B adapters
STAR mapping to repetitive elements, using hg19 Repbase sequences:
STAR --alignEndsType EndToEnd --outBAMcompression 10 --outFilterMultimapNmax 30 --outFilterMultimapScoreRange 1
--outFilterScoreMin 10 --outFilterType BySJout --outReadsUnmapped Fastx --outSAMattrRGline ID:foo --outSAMattributes All --outSAMmode Full --outSAMtype BAM Unsorted --outSAMunmapped Within --runMode alignReads
STAR mapping to Gencode release 19:
STAR --runMode alignReads --outStd BAM_Unsorted --outReadsUnmapped FastX --outSAMtype BAM Unsorted --outSAMattributes All --outSAMunmapped Within --outSAMattrRGline ID:foo --outFilterType BySJout --outFilterMultimapNmax 1 --outFilterMultimapScoreRange 1 --outFilterScoreMin 10 --outFilterScoreMinOverLread 0.3 --outFilterMatchNminOverLread 0.3
PCR duplicates were removed with barcodecollapsepe.py provided by (Van Nostrand et al., 2018).
Samtools was used to generate bam files containing R2 only, using the following parameters:
samtools view -f 128 -b
Peaks were identified with and normalized vs the size-matched input control as described (Van Nostrand et al., 2016), with the following parameters:
clipper --species hg19 --save-pickle
samtools view -cF 4
Input normalization was preformed with the perl script provided by (Van Nostrand et al., 2018):
overlap_peakfi_with_bam_PE.pl
Assembly: hg19
Supplementary files format and content: Bed files of eCLIP peaks called by Clipper
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| Submission date |
Jun 14, 2022 |
| Last update date |
May 09, 2023 |
| Contact name |
J. Robert Hogg |
| E-mail(s) |
j.hogg@nih.gov
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| Organization name |
National Heart, Lung, and Blood Institute
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| Department |
Biochemistry and Biophysics Center
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| Street address |
50 South Drive, Room 2341
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL21290 |
| Series (2) |
| GSE206059 |
A network of DZF proteins controls alternative splicing regulation and fidelity [eCLIP-seq] |
| GSE206061 |
A network of DZF proteins controls alternative splicing regulation and fidelity. |
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| Relations |
| BioSample |
SAMN29024432 |
| SRA |
SRX15696403 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6241094_ZFR-SMI-1_peaks.bed.gz |
1.3 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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