|
| Status |
Public on May 09, 2021 |
| Title |
HEK293T, RBFOX2 rep1 clip |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
rip antibody: anti-HA tag antibody
antibody manufacturer: Abcam
antibody catalog #: ab9110
adapter: InvRNA3
|
| Treatment protocol |
HEK293 cells were UV cross-linked with 600mJ and processed through the eCLIP protocol as previously described (van Nostrand et al, 2016). Anti-HA tag antibody was employed for immunoprecipitation.
|
| Growth protocol |
HEK293 cells were maintained in DMEM (4.5 g/L D-glucose) supplemented with 10% FBS (Gibco) at 37o C with 5% CO2. Cells were periodically passaged once at 70-90% confluency by dissociating with TrypLE Express Enzyme (Gibco) at a ratio of 1:10
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Biological replicates (according to ENCODE requirements) of 20 million cells were treated with 600mJ of UV using the Stratalinker 2400, harvested in ice cold PBS and pellets flash frozen in liquid nitrogen and stored in -80 degrees until ready to IP with TIA1 bound Dynabeads Subsequent protocol exactly as detailed in Van Nostrand et al 2016. Sequencing was performed on Illumina Hi Seq single end reads and data processed through Dr. Yeo's eCLIP pipeline.
Libraries were constructed as per the eCLIP protocol using Illumina 50x forward and 70x reverse primers.
eCLIP
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
PRBF_1.vs.PRBF_2.bed
|
| Data processing |
Sequenced reads were reformatted to include randomers in read headers with umi_tools (1.0.0). Args: --random-seed 1 --bc-pattern NNNNNNNNNN
Reads were then trimmed with cutadapt (1.14). Args: --match-read-wildcards -O 1 --times 1 -e 0.1 --quality-cutoff 6 -m 18 -a InvRNA*.fasta (fasta sequences can be found at: https://github.com/YeoLab/eclip/tree/master/example/inputs/)
Reads were then trimmed once more with cutadapt (1.14) to remove double-ligation events. Args: --match-read-wildcards -O 5 --times 1 -e 0.1 --quality-cutoff 6 -m 18 -a InvRNA*.fasta (fasta sequences can be found at: https://github.com/YeoLab/eclip/tree/master/example/inputs/)
Trimmed reads were then mapped with STAR (2.4.0i) against a human-specific repeat element database (RepBase 18.05). Args: --runThreadN 16 \ --genomeDir human_repbase \ --readFilesIn path/to/read1 \ --outFileNamePrefix out_prefix \ --outReadsUnmapped Fastx \ --outSAMtype BAM Unsorted \ --outSAMattributes All \ --outSAMunmapped Within \ --outSAMattrRGline ID:foo \ --outFilterType BySJout \ --outFilterMultimapNmax 30 \ --outFilterMultimapScoreRange 1 \ --outFilterScoreMin 10 \ --alignEndsType EndToEnd
Unmapped reads filtered of repeat elements were then mapped with STAR (2.4.0i) against a human genome (hg19). Args: --runThreadN 16 \ --genomeDir genomedir \ --readFilesIn /path/to/read1 \ --outFileNamePrefix out_prefix \ --outReadsUnmapped Fastx \ --outSAMtype BAM Unsorted \ --outSAMattributes All \ --outSAMunmapped Within \ --outSAMattrRGline ID:foo \ --outFilterType BySJout \ --outFilterMultimapNmax 1 \ --outFilterMultimapScoreRange 1 \ --outFilterScoreMin 10 \ --alignEndsType EndToEnd
Aligned reads were sorted with samtools (1.6)
Sorted reads were collapsed with umi_tools (1.0.0). Args: --random-seed 1 --method unique
BAM files were used to identify peak clusters with Clipper (1.2.2). Args: --species hg19 --bam path/to/input.bam --timeout 3600000 --maxgenes 1000000 --save-pickle --outfile path/to/output.bam
Peak clusters were normalized using BAM files for IP against BAM files for INPUT with peaksnormalize.pl (overlap_peakfi_with_bam_PE.pl), included in eclip 0.1.5+.
Overlapping normalized peak regions were merged with compress_l2foldenrpeakfi_for_replicate_overlapping_bedformat.pl, included within eclip-0.1.5+
Normalized peak (compressed.bed) files were ranked by entropy score (make_informationcontent_from_peaks.pl included within the merge_peaks pipeline) and used as inputs to IDR (2.0.2) to determine reproducible peaks.
Genome_build: hg19
Supplementary_files_format_and_content: BigWig, BED
|
| |
|
| Submission date |
Feb 08, 2021 |
| Last update date |
May 09, 2021 |
| Contact name |
Gene Yeo |
| E-mail(s) |
geneyeo@ucsd.edu
|
| Organization name |
UCSD
|
| Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE155729 |
Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes |
| GSE166402 |
Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [STAMP_new_eclip] |
|
| Relations |
| BioSample |
SAMN17838286 |
| SRA |
SRX10055402 |
