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| Status |
Public on Dec 20, 2021 |
| Title |
rpb1-T,FSP2, MNase-seq |
| Sample type |
SRA |
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| Source name |
Whole yeast cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
growth protocol: Rich medium, 30°, log phase
genotype: rpb1-T,FSP2
sample: 9930-1-3 #2
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| Growth protocol |
Cultures were grown to logarithmic phase in rich medium at 30° C, treated with 1% formaldehyde for 20 minutes, then harvested by centrigugation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Glycine (final 0.2 M) was added to quench crosslinking and cells were washed with 1X Tris-buffered saline. Cell walls were removed with 500 U lyticase, 37° 20 minutes, or until >90% of cells were spheroplasted and collected by centrifugation. Cells were lysed in MNase digestion buffer and 10 U MNase, then incubated at 37° for 15 minutes, and the reaction stopped, diluted, and crosslinks reversed by incubating 5 hours at 65°. DNA was precipitated with ethanol, then treated with RNase A, followed by Proteinase K digestion and purification on Qiagen Qiaquick columns.
Sequencing libraries were constructed using NEBNext® Multiplex Oligos (Index Primers Set 1 and Set 2) and NEBNext® ChIP-seq Library Prep Reagent Set for Illumina. Ligated samples were subjected to 50 bp paired-end sequencing in an Illumina NovaSeq 6000 sequencer.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
MNase resistant fraction
rpb1-T-FSP.normal.bw
rpb1-T-FSP.skinny.bw
rpb1-T-FSP.subnuc.bw
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| Data processing |
Reads were aligned to the yeast genome version SacCer3 using Novocraft novoalign version 3.7.1, with adapter trimming, allowing one random multi-mapping alignment.
Duplicate alignments were randomly sub-sampled to a level of 40% relative to non-duplicate reads using bam_partial_dedup, version 1.7, https://github.com/tjparnell/HCI-Scripts/tree/master/BamFile.
Fragment coverage was generated using bam2wig version 1.52 (https://github.com/tjparnell/biotoolbox) with an extension of 150 bp, skipping chrMito and high-copy number features including telomeric and rDNA loci, depth normalized to Reads Per Million, averaging between replicates, and converting to bigWig format.
Genome_build: SacCer3
Supplementary_files_format_and_content: bigWig files representing the nucleosomal fragment coverage (140-170 bp) or subnucleosomal fragments (90-120 bp), normalized to Reads Per Million, and averaged between replicates were generated.
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| Submission date |
Nov 29, 2021 |
| Last update date |
Dec 20, 2021 |
| Contact name |
Tim Formosa |
| E-mail(s) |
[email protected]
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| Phone |
8015815435
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| Organization name |
University of Utah School of Medicine
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| Department |
Biochemistry
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| Street address |
15 N Medical Dr East RM 4100
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| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112-5650 |
| Country |
USA |
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| Platform ID |
GPL27812 |
| Series (2) |
| GSE184955 |
Spt6-tSH2 domain and Rpb1 |
| GSE189831 |
The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II [MNase-seq] |
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| Relations |
| BioSample |
SAMN23496231 |
| SRA |
SRX13257140 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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