|
| Status |
Public on Jan 08, 2020 |
| Title |
spt3del_input_A |
| Sample type |
SRA |
| |
|
| Source name |
whole organism
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: SHY1187
genotype: mat alpha delta ade2::hisG his3 delta 200 leu2 delta 0 lys2 delta 0 met15 delta 0 trp1 delta 63 ura3 delta 0 RPB3 - Flag3 :: KanMX spt3 delta::NAT MX
treatment: 6 minute RNA labeling with 4-tU
growth: 30 degrees in synthetic medium to mid-log phase
|
| Growth protocol |
30 degrees in synthetic medium to mid-log phase
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq was performed similarly as described (Rodriguez et al., 2014; PMID: 25271716 ) with following modifications. Zirconia/silica beads were used for bead beating. Chromatin was sonicated for three rounds of 15 min. S. pombe (strain 972h) chromatin used as a spike-in was prepared the same way as S. cerevisiae samples (including cross linking, bead beating and sonication) and kindly supplied by Toshi Tsukiyama. Each IP reaction contained 1 ug of S. cerevisiae chromatin, 10 ng of S. pombe chromatin and 20 ul of Protein G dynabeads (Invitrogen, #10004D) conjugated with 4 ul of anti-H3K18Ac antibody (EMD Milipore, #07-354) and the final volume was brought to 500 ul with FA buffer. 25 ul of the S. cerevisiae/S. pombe chromatin mix was transferred to a separate tube as an ?input? sample before addition of beads and combined with 25 ul of the stop buffer. Chromatin-antibody complexes were eluted with two 25 ul volumes of stop buffer. IP and input samples were processed together from this point. After Proteinase K digestion DNA was purified by phenol extraction (two step extraction for input samples) followed by ethanol precipitation in the presence of 20 ug glycogen. Pelleted DNA was washed with 70% ethanol and resuspended in 15 ul 10 mM Tris-HCl (pH 7.5).
Sequencing libraries were prepared similarly as described (Warfield et al., 2017; PMID: 28918900) with few modifications. 2 ng of DNA was used as an input. Final adapter concentration during ligation was 6.5 nM. 15 cycles were used for library amplification.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
transcriptomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Paired-end sequencing reads were aligned to S. cerevisiae reference genome (sacCer3) and S. pombe reference genome (ASM294v2.20) with Bowtie2 using optional arguments “-I 10 -X 700 --local --very-sensitive-local --no-unal --no-mixed --no-discordant”.
For each base pair in the genome, we counted the number of paired-end fragments aligned over it.
Signal per base pair was normalized by the number of all S. pombe reads mapped for the sample, multiplied by 10000 (arbitrarily chosen number) and multiplied by the ratio of S. pombe to S. cerevisiae reads in the corresponding input sample.
Genome_build: sacCer3 (S. cerevisiae), ASM294v2.20 (S. pombe)
Supplementary_files_format_and_content: Processed data is provided in a wig format and contains normalized signal per base pair.
|
| |
|
| Submission date |
Dec 17, 2019 |
| Last update date |
Jan 08, 2020 |
| Contact name |
Rafal Donczew |
| E-mail(s) |
Rafal-donczew@omrf.org
|
| Organization name |
Oklahoma Medical Research Foundation
|
| Department |
Cell Cycle and Cancer Biology
|
| Lab |
Hahn Lab
|
| Street address |
825 NE 13th St
|
| City |
Oklahoma City |
| State/province |
OK |
| ZIP/Postal code |
73104 |
| Country |
USA |
| |
|
| Platform ID |
GPL17342 |
| Series (2) |
| GSE142122 |
Two separate roles for the transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA |
| GSE142183 |
Two separate roles for the transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA [ChIP-Seq] |
|
| Relations |
| BioSample |
SAMN13616495 |
| SRA |
SRX7401838 |
