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| Status |
Public on May 28, 2020 |
| Title |
wild-type S2 cells, TAF1 dsRNA knockdown, Replicate 2 |
| Sample type |
SRA |
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| Source name |
wild type S2
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: wild type S2
cell type: late stage embryonic cells
treatment: TAF1 dsRNA knockdown
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| Treatment protocol |
The method used was adapted from [Clift/Schuh Cell 2017]. HCT116 cells cultured in McCoy’s 5A medium were grown to approximately 70% confluency. Media was aspirated off, and the cells were washed with PBS. 2ml of trypsin per plate were used to harvest adherent cells, after which an equal volume of Opti-MEM was added to each plate to neutralize the trypsin. Cells were combined in a 50ml centrifuge tube and spun down at 2,000xg for 5 minutes, then washed in PBS and spun down again at 2,000xg for 5 minutes. Cells were counted using a hemocytometer and diluted to 25 million cells/mL. 100 µl reactions were prepared, and cells were re-suspended in Buffer R and anti-TAF1 C413 antibody. A pulse only control was prepared, which consisted of cells suspended only in Buffer R. Transfections were performed using the Neon Transfection Kit (1530V, 1ms width, 1 pulse). Transfected cells were then pipetted into 1 mL of Opti-MEM in a 35mm dish and incubated at 37˚C for 1 hour. The Opti-Mem media (containing some suspended cells) was then pipetted off and saved. 500 µl of PBS was added to the cells on the plates, which were then harvested and centrifuged at 6,000xg for 5 minutes. Supernatant was aspirated off, and cell nuclei were subsequently isolated. For D. melanogaster Schneider line 2 (S2) cells RNAi was performed as described [Clemens, 2000] using 20-40 µg dsRNA. Cells were incubated with dsRNA for 2.5 d.
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| Growth protocol |
HCT116 cells were grown in McCoy's media (Gibco, 16600082) with Gibco 100x Antibiotic-Antimycotic (Fisher Sci, 15240062) penicillin-streptomycin and 10% fetal bovine serum (FBS) supplementation. Drosophila cell culture and RNAi. D. melanogaster Schneider line 2 (S2) cells were maintained at 25°C in Schneider‘s medium containing 10% (vol/vol) Fetalplex (Gemini), 100 units/mL penicillin, and 0.1 mg/mL streptomycin. RNAi was performed as described [Clemens, 2000] using 20-40 µg dsRNA. Cells were incubated with dsRNA for 2.5 d.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclear run-on experiments were performed as described previously (Mahat, D. B., Kwak, H., Booth, G. T., Jonkers, I. H., Danko, C. G., Patel, R. K., et al. (2016). Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nature Protocols, 11(8), 1455–1476. http://doi.org/10.1093/bioinformatics/btq033) with the following modification. The final concentration of non-biotinylated CTP was raised from 0.25uM to 25uM, and the final library clean up and size selection was accomplished using 1X AMPure XP (Beckman).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
nascent RNA
nascent RNA (PRO-seq) from S2 cells
counts_s2.txt
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| Data processing |
processing of all sequencing data was performed using the NascentFlow Pipeline (doi: 10.17605/OSF.IO/NDHJ2)
Genome_build: GRCh38 for HCT116 cells, DM6 for S2 cells
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| Submission date |
Jun 14, 2019 |
| Last update date |
May 28, 2020 |
| Contact name |
Dylan J Taatjes |
| E-mail(s) |
[email protected]
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| Phone |
303 492-6929
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| Organization name |
University of Colorado Boulder
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| Department |
Biochemistry
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| Lab |
Taatjes Lab
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| Street address |
Campus Box 596
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| City |
Boulder |
| State/province |
CO |
| ZIP/Postal code |
80303-0596 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (1) |
| GSE132764 |
TFIID enables RNA polymerase II promoter-proximal pausing |
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| Relations |
| BioSample |
SAMN12059910 |
| SRA |
SRX6073177 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3891573_Taf_2_S5_R1_001.bedGraph.gz |
63.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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