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Sample GSM6049608 Query DataSets for GSM6049608
Status Public on Nov 19, 2022
Title SMI-Rep2
Sample type SRA
 
Source name iSLK-BAC16 (WT)
Organisms Homo sapiens; Human gammaherpesvirus 8
Characteristics cell type: iSLK cells
virus: BAC16 infectious clone
treatment: 1mM Sodium butyrate + 1 ug/mL doxycycline (24hr)
eclip antibody: No antibody (size-matched input)
Treatment protocol Cells were treated with 400mJ/cm2
Growth protocol iSLK-BAC16 (WT) cells were grown in DMEM supplemented with 10% Tet-free FBS. Cells at ~70% confluency were treated with 1mM sodium butyrate and 1 ug/mL doxycycline for 24 hours. Twenty million cells were harveested per sample.
Extracted molecule total RNA
Extraction protocol Cells were lysed with eCLIP lysis buffer (Eclipse Bio) at 20 million cells/ml, snap frozen in liquid nitrogen, and sent to Eclips Bio where the immunoprecipitation, library prep, and sequencing were performed. All folowed the methodology of van Nostrand et al. (2016; Nature Methods)
This was performed by a third party, Eclipse Bio. No modifications were made to the protocol of Van Nostrand et al (2016; Nature Methods).
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2500
 
Description RNA (human and KSHV)
Data processing UMIs were removed using umi_tools v0.5.1: umi_tools extract –-stdin=045IP13_S37.fastq.gz --bc-pattern=NNNNNNNNNN --log=045IP13_S37.processed.log --stdout 045IP13_S37.demultiplex.fastq.gz
Read quality was checked with Fast QC v.0.11.9: fastqc 045IP13_S37.demultiplex.fastq.gz
Reads were trimmed using two rounds of Cutadapt v3.2 to trim 3´ adaptors and to control for double ligation events: cutadapt --match-read-wildcards --times 1 -e 0.1 -O 1 --quality-cutoff 6 -m 18 -a AGATCGGAAGAGCAC -a GATCGGAAGAGCACA -a ATCGGAAGAGCACAC -a TCGGAAGAGCACACG -a CGGAAGAGCACACGT -a GGAAGAGCACACGTC -a GAAGAGCACACGTCT -a AAGAGCACACGTCTG -a AGAGCACACGTCTGA -a GAGCACACGTCTGAA -a AGCACACGTCTGAAC -a GCACACGTCTGAACT -a CACACGTCTGAACTC -a ACACGTCTGAACTCC -a CACGTCTGAACTCCA -a ACGTCTGAACTCCAG -a CGTCTGAACTCCAGT -a GTCTGAACTCCAGTC -a TCTGAACTCCAGTCA -a CTGAACTCCAGTCAC -o 045IP13_S37.adapterTrim.fastq.gz 045IP13_S37.demultiplex.fastq.gz > 045IP13_S37.adapterTrim.metrics and cutadapt --match-read-wildcards --times 1 -e 0.1 -O 5 --quality-cutoff 6 -m 18 -a AGATCGGAAGAGCAC -a GATCGGAAGAGCACA -a ATCGGAAGAGCACAC -a TCGGAAGAGCACACG -a CGGAAGAGCACACGT -a GGAAGAGCACACGTC -a GAAGAGCACACGTCT -a AAGAGCACACGTCTG -a AGAGCACACGTCTGA -a GAGCACACGTCTGAA -a AGCACACGTCTGAAC -a GCACACGTCTGAACT -a CACACGTCTGAACTC -a ACACGTCTGAACTCC -a CACGTCTGAACTCCA -a ACGTCTGAACTCCAG -a CGTCTGAACTCCAGT -a GTCTGAACTCCAGTC -a TCTGAACTCCAGTCA -a CTGAACTCCAGTCAC -o 045IP13_S37.adapterTrim.round2.fastq.gz 045IP13_S37.adapterTrim.fastq.gz > 045IP13_S37.adapterTrim.round2.metrics
Repetitive elements were removed using STAR v. 2.7.7a: STAR --runMode alignReads --runThreadN 8 --genomeDir repetitive_elements/genome/ --readFilesIn 045IP13_S37.adapterTrim.round2.fastq.gz --outSAMunmapped Within --outFilterMultimapNmax 30 --outFilterMultimapScoreRange --outFileNamePrefix 045IP13_S37.adapterTrim.round2.rep.bam --outSAMattributes All --readFilesCommand gunzip -c --outStd BAM_Unsorted --outSAMtype BAM Unsorted --outFilterType BySJout --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --alignEndsType EndToEnd > 045IP13_S37.adapterTrim.round2.rep.bam
Read quality after repetitive element removal was rechecked using Fast QC v. 0.11.9: fastqc 045IP13_S37.adapterTrim.round2.rep.bamUnmapped.out.mate1
Reads were mapped using STAR V2.7.7a: STAR --runMode alignReads --runThreadN 8 --genomeDir genome/ --readFilesIn 045IP13_S37.adapterTrim.round2.rep.bamUnmapped.out.mate1 --outSAMunmapped Within --outFilterMultimapNmax 1 --outFilterMultimapScoreRange 1 --outFileNamePrefix 045IP13_S37.adapterTrim.round2.rmRep.bam --outSAMattributes All --outStd BAM_Unsorted --outSAMtype BAM Unsorted --outFilterType BySJout --outReadsUnmapped Fastx --outFilterScoreMin 10 --outSAMattrRGline ID:foo --alignEndsType EndToEnd > 045IP13_S37.adapterTrim.round2.rmRep.bam
Resulting bam files were sorted and indexed using Samtools v. 1.9: samtools sort 045IP13_S37.adapterTrim.round2.rmRep.bam -o 045IP13_S37.adapterTrim.round2.rmRep.sorted.bam and samtools index 045IP13_S37.adapterTrim.round2.rmRep.sorted.bam
PCR duplicates were removed using Umi_tools dedup v0.5.1: umi_tools dedup --method=unique -I 045IP13_S37.adapterTrim.round2.rmRep.sorted.bam --log=045IP13_S37.dedup.log -S 045IP13_S37.adapterTrim.round2.rmRep.sorted.rmDup.bam
Resulting bam files were sorted and indexed using Samtools v. 1.9: samtools sort 045IP13_S37.adapterTrim.round2.rmRep.sorted.rmDup.bam -o 045IP13_S37.adapterTrim.round2.rmRep.sorted.rmDup.sorted.bam and samtools index 045IP13_S37.adapterTrim.round2.rmRep.sorted.rmDup.sorted.bam
Assembly: UCSC version GRCh37/hg19 with GQ994935.1
Supplementary files format and content: bigwig
 
Submission date Apr 19, 2022
Last update date Nov 19, 2022
Contact name Nicholas K Conrad
E-mail(s) nicholas.conrad@utsouthwestern.edu
Phone 2146480795
Organization name UT Southwestern Medical Center
Department Microbiology
Street address 6000 Harry Hines Blvd
City Dallas
State/province TX
ZIP/Postal code 75063
Country USA
 
Platform ID GPL24095
Series (2)
GSE201045 The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [eCLIP]
GSE201046 The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication
Relations
BioSample SAMN27631646
SRA SRX14917507

Supplementary file Size Download File type/resource
GSM6049608_045inp14_S34.CombinedID.merged.r2.norm.neg.bw 3.7 Mb (ftp)(http) BW
GSM6049608_045inp14_S34.CombinedID.merged.r2.norm.pos.bw 4.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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