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| Status |
Public on Oct 02, 2022 |
| Title |
PRO-seq, Rhesus LCL male, rmIFNa2, 1 hour, rep 1 |
| Sample type |
SRA |
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| Source name |
Mm 290-96
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| Organism |
Macaca mulatta |
| Characteristics |
Sex: Male
cell line: Mm 290-96
cell type: Lymphoblastoid cell line
genotype: WT
treatment: rmIFNa2
time: 1 hour
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| Treatment protocol |
48 hours prior to the IFN-α2 treatments, the cell cultures were switched to a similar media without antibiotics. To treat the LCLs with human IFN-α2, 10 ug of lyophilized purified protein obtained from Proteintech (Ref. HZ-1066 Lot. 0615-01) was resuspended in 100 uL of 0.1% BSA PBS, to yield a stock at 1.25x106 units/mL. The rhesus IFN-α2 was obtained from PBL Assay Science (Ref. 16105-1 Lot. 6987R) and already came resuspended in 0.1% BSA PBS at 4.03x106 units/mL. The LCLs were treated for 1 hour with either 100 units/mL of human IFN-α2, or with 100 units/mL of rhesus IFN-α2, or with an equal volume of 0.1% BSA PBS for the control untreated samples.
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| Growth protocol |
The human and rhesus LCLs were cultured in RPMI-1640 media (Gibco 72400-047) using 15% FBS (Gibco 10437-028) and 100 units/mL Penicillin-Streptomycin (Gibco 15140-122) in vent-cap T-25 flasks (Corning 430639), and kept at a confluency between 400,000 cells/mL to 800,000 cells/mL during cell culture.
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| Extracted molecule |
total RNA |
| Extraction protocol |
PRO-seq datasets were prepared as described in Mahat 2016 (https://doi.org/10.1038/nprot.2016.086). Briefly, between 8 to 12 million nuclei per dataset were used for the PRO-seq transcription run-on using a mixture of rNTP and Biotin-11-CTP (PerkinElmer NEL542001EA). Total RNA was extracted using a phenol/chloroform precipitation. Isolated RNA was fragmented using base hydrolysis with NaOH. Biotinylated fragmented nascent transcripts were isolated using a first streptavidin Dynabeads M-280 (Invitrogen 11206D) pull down, and the VRA3 RNA adaptor was ligated at their 3’ end. A second streptavidin bead pull down was performed, followed by the enzymatic modifications of the RNA fragment 5’ ends with a pyrophosphohydrolase and a polynucleotide kinase, and the VRA5 RNA adaptor was ligated at their fixed 5’ ends. A third streptavidin bead pull down was performed, followed by the reverse transcription of the resulting adaptor-ligated libraries. The libraries were cleaned up with AMPure XP beads (Beckman Coulter A63881). Then, the libraries were amplified using 13 PCR cycles, and cleaned up again with another round of AMPure XP beads. The resulting library concentrations were measured with the Qubit dsDNA high sensitivity assay (Invitrogen Q32851), and their size distributions assessed using the Agilent High Sensitivity D1000 ScreenTape. PRO-seq datasets were sequenced using an Illumina NextSeq as single-end 75 bp long reads.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Base calls and demultiplexing was done using Bcl2Fastq2 (v2.2.0).
Read quality and adapter trimming was done using BBMap (v38.05) bbduk with options ktrim=r qtrim=10 k=23 mink=11 hdist=1 maq=10 minlen=25 literal=AAAAAAAAAAAAAAAAAAAAAAA ref= The FASTA file containing common adapters found in https://github.com/Dowell-Lab/Nascent-Flow/blob/master/bin/adapters.fa.
Mapping was done usisng HISAT2 (v2.1.0) with options --very-sensitive --no-spliced-alignment.
SAM to BAM conversion was done using Samtools (v1.8).
Bidirectional loci were determined using Tfit and dREG as described in the Nextflow pipeline https://github.com/Dowell-Lab/Bidirectional-Flow. Tfit calls were obtained by first using the Tfit bidir module to call prelim regions. Added 3 kb-wide TSS regions to the prelim file and removed any part of the prelim regions that overlap with the TSS regions, cut the prelim regions that are > 10 kb down to equal size regions that have some overlap, coverage filtered to keep prelim regions having > 9 mapped reads. Finally, used the filtered/diced prelim regions on the Tfit model module to obtain Tfit calls. dREG calls were filtered as having FDR < 0.05, merged if within 20bp of each other, and having > 9 mapped reads.
Bigwigs were obtained using deepTools (v3.0.1) bamCoverage with options --binSize 1 --normalizeUsing RPKM --filterRNAstrand reverse (for pos file) or forward (for neg file) --scaleFactor 1 (for pos file) or -1 (for neg file).
Assembly: Homo sapiens datasets mapped to hg38, Macaca mulatta datasets mapped to rheMac10.
Supplementary files format and content: Files *.tfit.bed are 8-column BED files containing the interval of each detected bidirectional transcription locus. 1=Chr, 2=Start, 3=End, 4=Tfit Bidirectional Model Stats, 5-8= bedtools coverage interval report
Supplementary files format and content: Files *.dreg.bed are 7-column BED files containing the interval of each detected bidirectional transcription locus. 1=Chr, 2=Start, 3=End, 4-7= bedtools coverage interval report
Library strategy: PRO-seq
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| Submission date |
Sep 27, 2022 |
| Last update date |
May 16, 2023 |
| Contact name |
Robin D. Dowell |
| E-mail(s) |
robin.dowell@colorado.edu
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| Phone |
303 492 8204
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| Organization name |
University of Colorado Boulder
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| Department |
Molecular, Cellular and Developmental Biology
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| Lab |
Dowell lab
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| Street address |
596 UCB
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| City |
Boulder |
| State/province |
CO |
| ZIP/Postal code |
80309 |
| Country |
USA |
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| Platform ID |
GPL21120 |
| Series (1) |
| GSE214304 |
Nascent transcription upon interferon-α2 stimulation on human and rhesus macaque lymphoblastoid cell lines |
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| Relations |
| BioSample |
SAMN31042169 |
| SRA |
SRX17724329 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6603483_PRO-rmIFNa2-Rhesus-M.dreg.bed.gz |
348.8 Kb |
(ftp)(http) |
BED |
| GSM6603483_PRO-rmIFNa2-Rhesus-M.neg.bw |
80.0 Mb |
(ftp)(http) |
BW |
| GSM6603483_PRO-rmIFNa2-Rhesus-M.pos.bw |
82.9 Mb |
(ftp)(http) |
BW |
| GSM6603483_PRO-rmIFNa2-Rhesus-M.tfit.bed.gz |
785.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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