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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 06, 2016 |
| Title |
RANGAP_eCLIP_Replicate2 |
| Sample type |
SRA |
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| Source name |
RANGAP_eCLIP_Replicate2
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| Organism |
Homo sapiens |
| Characteristics |
antibody: RANGAP1
antibody manufacturer: Bethyl
antibody catalog: A302-026A
cell line: 293T
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| Treatment protocol |
While plated, cells were crosslinked with UV (254 nm, 400 mJ/cm2). Cells were then isolated with cell scrapers, pelleted at 200g, and snap frozen.
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| Growth protocol |
293T cells were grown under standard conditions (DMEM + 10% FBS + Pen/Strep (100U / mL)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed in iCLIP lysis buffer, followed by limited digestion with RNase I (Ambion), immunoprecipitation of RBP-RNA complexes with a specific antibody of interest (Protein G sheep anti-rabbit Dynabeads), and stringent washes.
After dephosphorylation with FastAP (Thermo Fisher) and T4 PNK (NEB), a barcoded RNA adapter is ligated to the 3’ end (T4 RNA Ligase, NEB) (at this step, multiple replicates of the same RBP, or potentially RBPs of similar size and bound RNA amount, can be uniquely barcoded and pooled after ligation to simplify downstream steps - see Supplementary Fig. 2A). Ligations are performed on-bead (to allow washing away unincorporated adapter) in high concentration of PEG8000, which improves ligation efficiency to > 90%. Samples are then run on standard protein gels and transferred to nitrocellulose membranes, and a region 75 kDa (~150 nt of RNA) above the protein size is isolated and proteinase K (NEB) treated to isolate RNA. RNA is reverse transcribed with AffinityScript (Agilent), and treated with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides. A second DNA adapter (containing a random-mer of 5 (N5) or 10 (N10) random bases at the 5’ end) is then ligated to the cDNA fragment 3’ end (T4 RNA Ligase, NEB), performed with high concentration of PEG8000 (to improve ligation efficiency) and DMSO (to decrease inhibition of ligation due to secondary structure). After cleanup (Dynabeads MyOne Silane, ThermoFisher), an aliquot of each sample is first subjected to qPCR (to identify the proper number of PCR cycles), and then the remainder is PCR amplified (Q5, NEB) and size selected via agarose gel electrophoresis.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
KB7_RANGAP_4
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| Data processing |
Takes output from raw files. Run to trim off both 5’ and 3’ adapters on both reads. Command: quality-cutoff 6 -m 18 -a NNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -g CTTCCGATCTACAAGTT -g CTTCCGATCTTGGTCCT -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGT AGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT -o /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.fastq.gz -p /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.fastq.gz /full/path/to/files/file_R1.C01.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.metrics
Takes output from cutadapt round 1. Run to trim off the 3’ adapters on read 2, to control for double ligation events. Command: cutadapt -f fastq --match-read-wildcards --times 1 -e 0.1 -O 5 --quality-cutoff 6 -m 18 -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGTAGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT -o /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.fastq.gz -p /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.round2.fastq.gz /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.fastq.gz > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.metrics
Takes output from cutadapt round 2. Maps to human specific version of RepBase used to remove repetitive elements, helps control for spurious artifacts from rRNA (& other) repetitive reads. Command: STAR --runMode alignReads --runThreadN 16 --genomeDir /path/to/RepBase_human_database_file --genomeLoad LoadAndRemove --readFilesIn /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.round2.fastq.gz --outSAMunmapped Within --outFilterMultimapNmax 30 --outFilterMultimapScoreRange 1 --outFileNamePrefix /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bam --outSAMattributes All --readFilesCommand zcat --outStd BAM_Unsorted --outSAMtype BAM Unsorted --outFilterType BySJout --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --alignEndsType EndToEnd > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bam
Takes output from STAR rmRep. Maps unique reads to the human genome. Command: STAR --runMode alignReads --runThreadN 16 --genomeDir /path/to/STAR_database_file --genomeLoad LoadAndRemove --readFilesIn /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bamUnmapped.out.mate1 /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bamUnmapped.out.mate2 --outSAMunmapped Within --outFilterMultimapNmax 1 --outFilterMultimapScoreRange 1 --outFileNamePrefix /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.bam --outSAMattributes All --outStd BAM_Unsorted --outSAMtype BAM Unsorted --outFilterType BySJout --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --alignEndsType EndToEnd > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.bam
takes output from STAR genome mapping. Custom random-mer-aware script for PCR duplicate removal. Command: barcode_collapse_pe.py --bam /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.bam --out_file /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.bam --metrics_file /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.metrics
Takes output from barcode collapse PE. Sorts resulting bam file for use downstream. Command: java -Xmx2048m -XX:+UseParallelOldGC -XX:ParallelGCThreads=4 -XX:GCTimeLimit=50 -XX:GCHeapFreeLimit=10 -Djava.io.tmpdir=/full/path/to/files/.queue/tmp -cp /path/to/gatk/dist/Queue.jar net.sf.picard.sam.SortSam INPUT=/full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.bam TMP_DIR=/full/path/to/files/.queue/tmp OUTPUT=/full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam VALIDATION_STRINGENCY=SILENT SO=coordinate CREATE_INDEX=true
Takes output from sortSam, makes bam index for use downstream. Command: samtools index /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam.bai
Takes inputs from multiple final bam files. Merges the two technical replicates for further downstream analysis. Command: samtools merge /full/path/to/files/CombinedID.merged.bam /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam /full/path/to/files/file_R1.D08.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam
Takes output from sortSam, makes bam index for use downstream. Command: samtools index /full/path/to/files/CombinedID.merged.bam /full/path/to/files/CombinedID.merged.bam.bai
Takes output from sortSam. Only outputs the second read in each pair for use with single stranded peak caller. This is the final bam file to perform analysis on. Command: samtools view -hb -f 128 /full/path/to/files/CombinedID.merged.bam > /full/path/to/files/CombinedID.merged.r2.bam
Takes results from samtools view. Calls peaks on those files. Command: clipper -b /full/path/to/files/CombinedID.merged.r2.bam -s hg19 -o /full/path/to/files/CombinedID.merged.r2.peaks.bed --bonferroni --superlocal --threshold-method binomial --save-pickle
Genome_build: hg19
Supplementary_files_format_and_content: bed format, contains clusters of predicted RBP binding
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| Submission date |
Aug 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gene Yeo |
| E-mail(s) |
geneyeo@ucsd.edu
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| Organization name |
UCSD
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| Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE86035 |
SONAR discovers RNA binding proteins from analysis of large-scale protein-protein interactomes. |
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| Relations |
| BioSample |
SAMN05631168 |
| SRA |
SRX2052812 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2291983_KB6-7_02.basedon_KB6-7_02.peaks.l2inputnormnew.bed.compressed.bed.gz |
630.3 Kb |
(ftp)(http) |
BED |
| GSM2291983_KB7_RANGAP_4_RANGAP1.merged.r2.norm.neg.bw |
12.9 Mb |
(ftp)(http) |
BW |
| GSM2291983_KB7_RANGAP_4_RANGAP1.merged.r2.norm.pos.bw |
12.3 Mb |
(ftp)(http) |
BW |
| GSM2291983_KB7_RANGAP_4_RANGAP1.merged.r2.peaks.fixed.bb |
2.5 Mb |
(ftp)(http) |
BB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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