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| Status |
Public on Apr 06, 2022 |
| Title |
Nucleosome mapping in SNF2 depletion yeast cells |
| Sample type |
SRA |
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| Source name |
Yeast cell
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: MATa, can1, his3, leu2, ura2, hoprurs2 (nt-1298 to -272), tor1-1, fpr1::NAT, RPL13A-2XFKBP12::TRP1, SNF2: SNF2-FRB-TAP::kanMX, ho:CYC1corepr(7xtetO)-SV40NLS-M.CviPI:TDH3pr-TetR-SSN6:TDH3pr-rvTetR-VP16AD-Gal4AD::KlLEU2, CLN2:HOprurs2-swi5mut-NDFmotifs-GFP:CaURA3
treatment: 2hr 1ug/ml Rapamycin followed by 2hr 10ug/ml Doxycycline
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| Treatment protocol |
Yeast cells were treated with 1ug/ml of Rapamycin for 2 hour followed by 10ug/ml Doxycycline for 2 hours.
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| Growth protocol |
Yeast was grown in SCD-Ura-Met medium to OD660 0.2
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Yeast cells were digested by 100ug/ml of Zymolyase 100T at 37 degree and lysed by lysis buffer (0.28M EDTA pH8.0, 0.22M Tris-HCl pH8.0, and 2.2%SDS) at 65 degree. Genomics DNA was then extracted using phenol-cholorform-isoamly alcohol. Genomic DNA was treated with RNaseA, and cleaned up by Omega cycle pure kit.
EpiTect Fast DNA Bisulfite kit was used to bisulfite convert methylated genomic DNA. 1µg of DNA was used for each reaction; 2 cycles of 95degree for 5min and 60degree for 20min were used to convert DNA. Target synthetic libraries were then amplified by EpiMark Hot Start Taq DNA Polymerase and Turbo Cx Pfu. A 25ul PCR reaction contains 1ul of bisulfite-converted template, 0.5ul of each 10uM primer, 5ul of 5X EpiMark Taq buffer, 0.5ul of 10mM dNTPs, 0.125ul of EpiMark Hot Start Taq DNA Polymerase, and 0.05ul of Pfu Turbo Cx HotStart DNA polymerase. PCR thermal cycle is shown below: 1 cycle of 95 for 5min, 23cycles of 95degree for 25s, 55degree for 30s and 68degree for 90s, and 1cycle of 68degree for 5min. PCR reactions were concentrated using Amicon Ultra 30K filters and then cleaned up using SPRI beads.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Sequel |
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| Data processing |
Pacbio subreads with at least 3 passes were converted to consensus sequences using the ccs command.
CCS reads were aligned using MATLAB scripts based on global and local alignment. GC and other C in each reference sequence were converted to GY and T, respectively. We removed reads if 1) alignment accuracy is lower than 90%, 2) there is more than one mismatch in the synthetic oligo region (430-540 bp in the reference sequence that contains NDF motifs), 3) there is a mismatch in NDF motifs, or 4) methylation level too low (< four methylated GC in total).
Aligned DNA sequences were converted to numeric arrays. Methylated GC (GC in read) was converted to 1, unmethylated GC (GT in read) was converted to -1. Other nucleotides were converted to 0. Arrays with less than four 1 were removed. Arrays were piled up to generate a matrix.
Methylation level of each biosample was normalized to one of the WT sample (SRR18404682, sample ID:17R) to account for the variantion of M.CviPI expression.
In nucleosome prediction step, we assumed that the DNA is fully protected towards the nucleosome dyad but has some flexibility near the entry/exit sites. We correlated this pattern with the methylation measurement in a 147 bp window from 5’ to 3’ to get a “nucleosome penalty score” and placed nucleosomes at the local minimums. In scenarios where a nucleosome covers NDF motifs, we calculated the total methylation level in the “NDR region” (from -958 bp to -405 bp). If it is higher than 90% of that in the background sequences, we assumed that the nucleosome-like protection was generated by other complexes, and this nucleosome was removed. We then further optimized the nucleosome positions to remove the bias generated by 5’ to 3’ scanning. When a protected region has a length between mono- and di-nucleosomes, the nucleosome was set as 160 bp and placed at the center of the protected region. If this lengthy protection was adjacent to an NDF motif, we placed the nucleosome at the edge of this region away from the motif assuming that part of the protection was generated by the NDF, or factors recruited by the NDF.
Supplementary files format and content: Normalized and combined DNA methylation matrixes were storaged in txt files. In methylation matrix, 1, -1, and 0 represent methylated GC, unmethylated GC, and other nucleotide, respectively. Each txt file contains 169 matrixes which aligned to the169 sythetic oligoes in the library. Nucleosome prediction matrixes were storaged in csv files. In nucleosome matrix, each number represent the nucleosome occupancy at each position . Each csv file contains 169 matrixes which aligned to the169 sythetic oligoes in the library. The title of each matrix contains the sample name, sequence index, and the NDF name. For example, methylation_matrix_INO80_1_ABF1 means this methylation matrix was from INO80 depletion strain; the index of the sequence was 1; and this sequence containing ABF1 motif.
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| Submission date |
Mar 30, 2022 |
| Last update date |
Apr 06, 2022 |
| Contact name |
Lu Bai |
| E-mail(s) |
lub15@psu.edu
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| Phone |
8148634824
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| Organization name |
Penn State University
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| Street address |
406 South Frear
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| City |
UNIVERSITY PARK |
| State/province |
PA |
| ZIP/Postal code |
16802-1004 |
| Country |
USA |
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| Platform ID |
GPL32099 |
| Series (1) |
| GSE199812 |
Partitioned Usage of Chromatin Remodelers by Nucleosome Displacing Factors |
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| Relations |
| BioSample |
SAMN26856154 |
| BioSample |
SAMN26856153 |
| SRA |
SRX14538565 |
| SRA |
SRX14538564 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5987791_SNF2_met.txt.gz |
1.5 Mb |
(ftp)(http) |
TXT |
| GSM5987791_SNF2_nuc.csv.gz |
5.3 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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