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| Status |
Public on May 13, 2024 |
| Title |
Pol II NET-seq, CaCl2-treated, replicate 3 |
| Sample type |
SRA |
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| Source name |
CaCl2-treated
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell type: CaCl2-treated
genotype: RPB2-(HA)3-(His)7::HIS3Mx6
treatment: CaCl2-treated
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| Treatment protocol |
Immediately prior to the immunoprecipitation, frozen cell lysates were resuspended in lysis buffer (5 mL * grindate weight; previously described in Clarke et al, PNAS (2018)). For CaCl2 treated samples, a final concentration of 5 mM of CaCl2 was added, then samples were mixed thoroughly and incubated at 37ºC for 2 minutes. To quench the reaction, a final concentration of 5 mM of EDTA (pH 8.5) was added. For the MnCl2 treated samples, a final concentration of 10 mM MnCl2 was added and samples were mixed well. Samples were incubated on ice for 20 minutes, then the reaction was quenched with the addition of 5 mM EDTA (pH 8.5). Finally, samples were incubated on ice for 30 minutes.
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| Growth protocol |
Yeast were grown at 30ºC with nutation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell harvest, lysis, and RNA extraction were performed exactly the same as described in previous publications from our lab. To summarize, yeast cells were rapidly harvested via filtration and lysed under cryogenic conditions. Pol II was isolated via immunoprecipitation and an acidic phenol/chloroform extraction was used to extract total RNA.
NET-seq library construction was performed exactly as described in previous publications from our lab. To summarize, a UMI-containing DNA linker was ligated onto the 3' end of RNAs after the total RNA extraction. Next, reverse transcription was performed and resulting DNAs were circularized. Finally, circular DNAs were amplified, libraries were purified via PCRClean DX beads, and reads were sequenced with the Illumina NovaSeq 6000 machine.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
CaClC
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| Data processing |
*library strategy: NET-seq
Base-calling was conducted with bcl convert software.
Deduplication based on the UMI sequence was performed via fqtrim (version 0.9.7) with the "-C" option.
First trimming step was achieved via cutadapt (version 3.4) and the "-g -no-indels" parameters. Reads containing the 5' sequence 5'-AGNNNNNNNNTG-3' were isolated, and this sequence was removed.
Second trimming step was performed with cutadapt (version 3.4) and the "-a -no-indels" parameters. The 3' sequence 5'-CTGTAGGCACCAT-3' was removed from reads.
Reads were aligned to the Saccharomyces cerevisiae genome via STAR (version 2.7.1a). The STAR alignIntronMax parameter was set to 1.
Output BAM files from alignment were indexed using SAMtools (version 1.6).
The bamCoverage tool from the deepTools package was used to generate bigWig files, with parameters set to "--Offset 1 --normalizeUsing CPM".
The computeMatrix tool (scale-regions subtool) from the deepTools package with the parameters set to "-b 3000 -a 3000 --regionBodyLength 5000 --skipZeros" was used.
The plotProfile tool from the deepTools package was used to visualize data.
Assembly: S. cerevisiae R64-1-1
Supplementary files format and content: Processed data for each sample are included in an individual bigWig (.bw) file.
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| Submission date |
May 09, 2024 |
| Last update date |
May 13, 2024 |
| Contact name |
Abigail Huffines |
| E-mail(s) |
mcconaha@uab.edu
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| Organization name |
University of Alabama at Birmingham
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| Street address |
720 20th St S
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| City |
Birmingham |
| ZIP/Postal code |
35233 |
| Country |
USA |
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| Platform ID |
GPL27812 |
| Series (1) |
| GSE267082 |
The effect of metal cations on the capture of Pol II-derived nascent transcripts in NET-seq experiments |
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| Relations |
| BioSample |
SAMN41279057 |
| SRA |
SRX24505700 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8259519_CaClC_CPM.bw |
1.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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