|
| 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 |
