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| Status |
Public on Mar 09, 2020 |
| Title |
PRO-seq-13-NELFC-AID-FP |
| Sample type |
SRA |
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| Source name |
DLD-1
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Colorectal adenocarcinoma
genotype: NELF-C-AID OsTIR1
treatment: Flavopiridol
cell line: DLD-1
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| Treatment protocol |
Cells were treated with 500 µM Auxin (3-indole-acetic acid sodium salt, abcam), 1 µM Flavopiridol (Cayman), or 250 nM NVP-2 (MedChemExpress). Heat shock treatment at 42 ˚C for 1 h was carried out as described previously (Mahat et al, 2016)
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| Growth protocol |
DLD-1 cells were grown in DMEM (Corning) supplemented with 10% FBS (Corning), 1X GlutaMAX (Gibco), 2 µg/ml puromycin (Gibco), 100 U/ml Penicillin and 100 µg/ml Streptomycin (Gibco).
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
For ChIP-seq, 20-50 million cells were crosslinked with 1% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min at r.t. Spike-in mouse embryonic fibroblast fixed cells were added to samples as described previously (Orlando et al., 2014) for NELF-C, NELF-E, SPT5, Pol II CTD Ser2P, Pol II CTD Ser5P, XRN2, DCP2 ChIP-seq in NELF-C-AID cells. Chromatin was sonicated with the Covaris E220 for 4 min with 10% duty cycle, 140 peak intensity power, 200 cycles per burst. Immunoprecipitations were carried out with antibodies and Protein A/G-agarose beads (Santa Cruz) or Dynabeads Protein G (Invitrogen). PRO-seq sample preparation was performed according to the previously published protocol (Mahat et al., 2016b) with minor modifications. 10 million DLD-1 nuclei were mixed with spike-in Drosophila S2 nuclei. Nuclear run-on assay was performed with 4 biotinylated nucleotides (PerkinElmer). 5’ cap was removed by RppH (NEB). PRO-cap libraries were prepared as described previously (Kwak et al., 2013; Mahat et al., 2016b) with minor modifications. NELF-C-AID cells were treated with or without 500 µM auxin for 2 h, followed by nuclear extraction. ~10 million NELF-C-AID nuclei with 0.5 million spike-in drosophila S2 nuclei were subjected to nuclear run-on reaction.
For ChIP-seq, DNA libraries were prepared by the HTP Library Preparation Kit for Illimina (KAPA) and sequenced on the NextSeq 500 or NovaSeq 6000 (Illumina). For PRO-seq, adapter sequences previously described (Mahat et al., 2016) were used. DNA Libraries were size selected by PippinHT (Sage) or AMpure XP (Beckman Coulter) and sequenced on a NextSeq 500 (Illumina). For PRO-cap, PRO-seq adapter sequences were used instead of PRO-cap adapters. Libraries were then sequenced by NextSeq 500 in paired-end sequencing mode.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
PRO-seq
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| Data processing |
Library strategy: PRO-seq
For ChIP-seq, single-end raw reads trimmed by Trimmomatic 0.33 (Bolger et al., 2014) were aligned to a concatenated genome comprised of human hg19 and mouse mm9 assemblies, using bowtie 2.2.6 (Langmead and Salzberg, 2012) with --sensitive option. The aligned reads with MAPQ ≥ 30 were extended to 150 bp and read counts were normalized to total reads aligned to spike-in genome. Metaplots of ChIP-seq signals were generated using deepTools 3.0.0/3.1.1/3.1.2 computeMatrix, plotHeatmap, plotProfile (Ramírez et al., 2016). ChIP-seq peaks were called using MACS 2.1.0 (Zhang et al., 2008) with a q-value cutoff of 0.05. Peaks were annotated using HOMER 4.10 (Heinz et al., 2010).
For PRO-seq, low-quality bases and adapters from 3’ ends of reads were removed using cutadapt 1.14 (Martin, 2011) 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 --very-sensitive option (Langmead and Salzberg, 2012), to a concatenated genome comprised of human hg19 and fly dm3 assemblies. The 5’ ends of aligned reads with MAPQ ≥ 30 were taken by bedtools genomecov 2.25 (Quinlan and Hall, 2010) with -strand and -5 options, and strands of the 5’ ends of reads were then swapped. Read counts were normalized to total reads aligned to the spike-in genome. Protein coding transcripts were chosen from Ensembl version 75 database by R 3.3.3, according to the previously described protocol (Liang et al., 2018) with minor modifications. Briefly, we selected RefSeq-validated transcripts with TSSs that overlapping NELF-C ChIP-seq peaks. We filtered out transcripts that are ≤ 2 kb long and ≤ 2 kb away from the nearest genes. Next, we chose transcripts that have ≥ 2 rpm total PRO-seq coverage at the region between TSS – 100 bp and TSS + 300 bp, using 3 independent PRO-seq data in untreated NELF-C-AID cells. In these selected transcripts, the nucleotide position with maximum PRO-seq coverage in the region between TSS – 100 bp and TSS + 300 bp was assigned to the 1st pause region. Transcripts that have ≤ 0.2 rpm coverage at the 1st pause region nucleotide were excluded. The resulting transcripts with assigned the 1st pause regions were used for further analyses. The nucleotide positions with maximum PRO-seq coverage in 1h auxin-treated NELF-C-AID cells at the region from +10 to +500 bp downstream of the 1st pause region was assigned to the 2nd pause region. For Heat shock experiments, transcripts with ≥ 1.5-fold increase or decrease in PRO-seq coverage at gene bodies (the 1st pause regions +500 to +100kb or to transcription termination sites) upon heat shock were assigned to HS-upregulated or HS-downregulated genes, respectively. Heatmaps and metaplots of PRO-seq signals were generated by deepTools 3.0.0/3.1.1/3.1.2 computeMatrix, plotHeatmap, and plotProfile (Ramírez et al., 2016). Log2 fold change of PRO-seq signals at nucleotide resolution were calculated by deepTools 3.0.0/3.1.1/3.1.2 bigwigCompare with pseudocount 0.25 rpm.
For PRO-cap, raw reads were processed as described for PRO-seq above, requiring a read length of 18–36 bp. Processed paired-end reads were aligned to a concatenated genome comprised of hg19 and dm3 assemblies using bowtie2 with options (--very-sensitive -X 1000 --no-mixed --no-discordant --no-unal) (Langmead and Salzberg, 2012). The 5’ ends of aligned R2 reads with MAPQ ≥ 40 were taken by bedtools genomecov 2.25 (Quinlan and Hall, 2010) with -strand and -5 options. Read counts were normalized to total reads aligned to the spike-in genome. de novo transcription start sites were identified from PRO-cap data using HOMER 4.11 with findcsRNATSS option (Duttke et al., 2019; Heinz et al., 2010). TSS identified in untreated and auxin-treated samples were merged then defined as TSS regions. Distances between TSS in each sample were measured using HOMER 4.11 annotatePeaks. Raw PRO-cap reads were counted using HOMER 4.11 annotatePeaks. Read counts were normalized to spike-in counts using RUVseq with RUVg option (Risso et al., 2014) in R 3.3.3. Differential expression analysis was performed using DESeq2 (Love et al., 2014) and visualized in R 3.3.3. TSS with significant gain of PRO-cap signals upon NELF loss (adjusted p-value < 0.05) was defined as Cap Up TSS, while TSS with significant loss of PRO-cap signals upon NELF loss (adjusted p-value < 0.05) was defined as Cap Down TSS. Metaplots of PRO-cap and PRO-seq at Cap Up TSS or Cap Down TSS were generated using deepTools 3.1.1. For heatmaps of ChIP-seq and MNase-seq at TSS, TSS were first merged with gap of no longer than 600 bp to obtain TSS clusters, heatmaps were then generated at Cap Up TSS clusters and Cap Down TSS clusters.
Genome_build: hg19, dm3, mm9
Supplementary_files_format_and_content: bigWig files with normalized read counts were provided for all samples.
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| Submission date |
Feb 05, 2020 |
| Last update date |
Mar 09, 2020 |
| 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) |
| GSE144786 |
NELF regulates a promoter-proximal step distinct from RNA Pol II pause-release |
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| Relations |
| BioSample |
SAMN14001922 |
| SRA |
SRX7677757 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4296326_PRO-seq-13-NELFC-AID-FP.minus.bw |
22.6 Mb |
(ftp)(http) |
BW |
| GSM4296326_PRO-seq-13-NELFC-AID-FP.plus.bw |
23.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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