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| Status |
Public on Mar 23, 2023 |
| Title |
PRO-seq_DLD1_DMSO |
| Sample type |
SRA |
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| Source name |
colon
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| Organism |
Homo sapiens |
| Characteristics |
tissue: colon
cell line: DLD-1
cell type: colorectal adenocarcinoma
genotype: WT
treatment: DMSO
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| Treatment protocol |
ChIP-seq: HCT116 and DLD1 cells were treated with 20μM of iPAF1C or DMSO as a control. Knocking down PAF1 was achieved by transducing cells with a lenti-viral vector expressing shRNA against PAF1.
RNA-seq: 1million of DLD1_PAF1_AID cells were plated in a 6-well plate 24 hrs prior to treatment with 20μM iPAF1C versus DMSO or 500 μM auxin versus non-treated cells.
Heat shock of mammalian cells was performed using ∼70%–80% confluent DLD1 cells by 15 adding pre-heated (42°C) conditioned media collected from identically growing cells (Mahat et al., 2016b). The heat shock cells were incubated at 42°C for 1.5 hour.
J-Lat 5a8, 11.1, and 6.3 cell lines were plated in 96-well flat bottom plates at a density of 150,000 cells/250 μL supplemented RPMI. Cells were DMSO-treated or treated with iPAF1C (12.5 μM), JQ1 (1μM), PHA (0.5 μg/mL), and PMA (1 nM) for 48 hours.
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| Growth protocol |
DLD1 and HCT116 cells were maintained in DMEM supplemented with 10% FBS. J-LAT 5a8, 11.1, and 6.3 were cultured in RPMI-1640 supplemented with 5 mM HEPES, 50 μg/mL P/S, 5 mM sodium pyruvate, and 10% FBS. Cells were cultured at 37°C in humidified incubator with 5% CO2. For RNA interference, cells were infected with a lentivirus containing shRNA against PAF1 using 8 μg/ml polybrene for 24hrs followed by 2 μg/ml puromycin for 2 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of total RNA was performed using RNeasy Mini kit (Qiagen, Cat# 74106). Briefly, 1million of DLD1_PAF1_AID cells were plated in a 6-well plate 24 hrs prior to treatment with 20μM iPAF1C versus DMSO or 500 μM auxin versus non-treated cells.
ChIP-seq: ChIP-seq was performed according to a previously published protocol (Lee et al., 2006). For RNA Pol II, and PAF1 ChIP-seq, the chromatin was sonicated using Covaris E220 for 4 min using the following sonication conditions: 10% duty cycle, 140 peak intensity power, 200 cycles per burst. Successively, pulldown of chromatin was carried out overnight at 4°C using the specific antibodies (Rpb1 NTD (D8L4Y) rabbit mAb for RNA Pol II and PAF1 (D9G9X) Rabbit mAb). DNA libraries were prepared by the HTP Library Preparation Kit for Illumina (KAPA). RNA-seq: Extraction of total RNA was performed using RNeasy Mini kit (Qiagen, Cat# 74106). Briefly, 1million of DLD1_PAF1_AID cells were plated in a 6-well plate 24 hrs prior to treatment with 20μM iPAF1C versus DMSO or 500 μM auxin versus non-treated cells. For performing RNA-seq polyadenylated RNA was isolated using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, Cat# E7490L). This isolated poly(A)+ RNA was used to prepare libraries using NEBNext Ultra II Directional RNA Library Prep Kit (NEB, Cat# E7760L). PRO-seq: PRO-seq was performed according to the previously published protocol with minor modifications (Mahat et al., 2016a). Reverse transcription was performed with SuperScript III (ThermoFisher, Cat#18080044). cDNA was amplified using Phusion Hot Start II DNA polymerase (ThermoFisher, Cat#F549). DNA Libraries were purified using Beckman Coulter AMpure XP (Fisher Scientific, Cat#NC9959336).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Basecalls were performed using bcl2fastq v2.19 for Novaseq output.
ChIP-seq: low-quality bases were removed using Trimmomatic v0.33, with parameters TRAILING:30 MINLEN:20. Reads were aligned with bowtie v1.1.2 with parameters -m 1 -v 2, to human GRCh38 genome assembly with Ensembl gene annotation GRCh38 release 78. J-LAT 5a8 cells were aligned to the concatenated hg38/HIV-1 genome, using the same parameters as for the human samples. After alignment, HIV-1 reads were filtered out and normalized by the mapped read counts. Bigwig files were generated using an in-house R script utilizing bam2bw function, with a parameter to extend reads length -extLen=150 and scaled to reads-per-million (rpm).
RNA-seq:low-quality bases were removed using Trimmomatic v0.33, with parameters TRAILING:30 MINLEN:20. Reads were aligned to human GRCh38 genome assembly with Ensembl gene annotation GRCh38 release 78 using TopHat2 v2.1.0 (Kim et al., 2013), with parameters --no-novel-juncs --read-mismatches 2 --read-edit-dist 2 --max-multihits 20. Reads with non-primary alignment and low mapping quality were discarded. Reads were quantified using HTSeq v0.6.1.
PRO-seq: Low-quality bases and adapters from 30 ends of reads were removed using cutadapt 1.14 requiring a read length of 16–36 bp. Reads derived from ribosomal RNA were filtered out by mapping reads on human and fly ribosomal DNA. The remaining reads were aligned using bowtie 2.2.6 with option --very-sensitive, to a concatenated genome comprised of human hg38 (sample) and fly dm6 (spike-in) assemblies. The 5’ ends of aligned reads with MAPQ 30 were processed using bedtools genomecov v2.25.0 with paremeters -strand, -bg, and -5 to calculate coverage of 5’ ends. Read counts were normalized with total reads aligned to the spike-in genome (dm6), final bigwig files were scaled 10 to reads-per-million (rpm).
Assembly: hg38
Supplementary files format and content: bigWig files contain strand-specific, spike-in normalized PRO-seq signal.
Library strategy: PRO-seq
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| Submission date |
Aug 19, 2022 |
| Last update date |
Mar 23, 2023 |
| Contact name |
Ali Shilatifard |
| E-mail(s) |
[email protected]
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| Organization name |
Northwestern University Feinberg School of Medicine
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| Department |
Department of Biochemistry and Molecular Genetics
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| Lab |
Shilatifard Lab
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| Street address |
320 E Superior St
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE211651 |
Enhancing HIV-1 latency reversal through regulating the elongating RNA Pol II pause-release by a small-molecule disruptor of PAF1C |
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| Relations |
| BioSample |
SAMN30413340 |
| SRA |
SRX17152991 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6482060_PRO-seq_DLD1_DMSO.minus.bw |
64.4 Mb |
(ftp)(http) |
BW |
| GSM6482060_PRO-seq_DLD1_DMSO.plus.bw |
66.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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