|
| Status |
Public on Jan 17, 2024 |
| Title |
npd09 |
| Sample type |
SRA |
| |
|
| Source name |
haploid cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
tissue: haploid cells
cell line: S288C
genotype: W303-1A MATa ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100 RAD5+
treatment: M.SssI
treatment time: 60-180 min
|
| Treatment protocol |
25 µg of DNA, 20 µl RNase A of a 10 mg/ml stock, 10 µl M.SssI methyltransferase (20.000 U/ml), 601-nucleosome arrays (75-100 ng DNA) as an internal spike-in and cold 1x M.SssI buffer to a final volume of 397.5 µl was mixed and samples were incubated for 30-180 minutes.
|
| Growth protocol |
WT yeast strains were grown at 30°C in YPAD.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified by Phenol:Chloroform:Isoamylalcohol approach.
Library preparation of isolated DNA was performed with the SQK-LSK109 ligation sequencing kit (Oxford Nanopore Technologies) according the manufacturer’s protocol. For multiplexing up to 24 samples, native barcode expansion kits (EXP-NBD104/114) were used. Libraries were loaded onto MinION R9.4.1 flow cells (Oxford Nanopore Technologies) and run for 72 hrs using MinKNOW software (version 20.06.4) with disabled basecalling, barcoding and alignment. Output was saved in FAST5 format.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
MinION |
| |
|
| Description |
S288C_pFMP233_(25mer 601 WT); methylated by M.SssI
|
| Data processing |
Basecalling and demultiplexing were done using ONT guppy software by Oxford Nanopore Technologies (version 5.0.16).
Resulting fastq files were split per barcode by guppy_barcoder_out using the following parameters: –barcode_kits "EXP-NBD104 EXP-NBD114" --device auto.
FASTQ files were merged into a single file for each barcode.
Demultiplexed FASTQ files for each barcode were mapped to the SacCer3 (R64-1-1) reference genome by minimap2 (version 2.24), using minimap2 -ax map-ont.
Resulting alignments were further filtered, sorted and indexed by Samtools (version 1.9) using the following parameters: samtools view –F 2052 –q 30 –b –S; samtools sort –o; samtools index –b.
CpG methylation for each barcode were called by Nanopolish (version 0.13.2), using default parameters for ‘cpg’ motif with DNA sequences in FASTQ files, mapping results in BAM files and the same genome reference as was used for the sequence alignment as inputs.
Methylation states for each CpG, derived by Nanopolish, were defined by log likelihood using the following thresholds: methylated (≥ 2.5), unmethylated (≤ -2.5) and ambiguous (between -2.5 and 2.5).
Methylation rate was defined as a ratio of number of methylated sites per read to the total number of either methylated or unmethylated sites in the read. Reads longer than 1000 bp and containing methylation rates between 0.1 and 0.9 were selected for the further analysis in R.
Assembly: SacCer3
Library strategy: Fiber-Seq
|
| |
|
| Submission date |
Feb 07, 2023 |
| Last update date |
Jan 17, 2024 |
| Contact name |
Mark Boltengagen |
| E-mail(s) |
m.boltengagen@gmail.com
|
| Organization name |
Dresden Technical University
|
| Department |
Physiological Chemistry
|
| Lab |
Felix Müller-Planitz
|
| Street address |
Fiedlerstraße 42
|
| City |
Dresden |
| State/province |
Saxony |
| ZIP/Postal code |
01307 |
| Country |
Germany |
| |
|
| Platform ID |
GPL25739 |
| Series (1) |
| GSE224713 |
A single fiber view of the nucleosome organization of eukaryotic chromatin |
|
| Relations |
| BioSample |
SAMN33189372 |
| SRA |
SRX19490703 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7030158_meth_data_WT_TC1_120_filtered.tsv.gz |
26.4 Mb |
(ftp)(http) |
TSV |
| GSM7030158_meth_data_WT_TC1_180_filtered.tsv.gz |
33.0 Mb |
(ftp)(http) |
TSV |
| GSM7030158_meth_data_WT_TC1_60_filtered.tsv.gz |
22.3 Mb |
(ftp)(http) |
TSV |
| GSM7030158_meth_data_WT_TC5_120_filtered.tsv.gz |
26.9 Mb |
(ftp)(http) |
TSV |
| GSM7030158_meth_data_WT_TC5_180_filtered.tsv.gz |
42.0 Mb |
(ftp)(http) |
TSV |
| GSM7030158_read_stats_WT_TC1_120_filtered.tsv.gz |
1.5 Mb |
(ftp)(http) |
TSV |
| GSM7030158_read_stats_WT_TC1_180_filtered.tsv.gz |
2.1 Mb |
(ftp)(http) |
TSV |
| GSM7030158_read_stats_WT_TC1_60_filtered.tsv.gz |
1.2 Mb |
(ftp)(http) |
TSV |
| GSM7030158_read_stats_WT_TC5_120_filtered.tsv.gz |
2.0 Mb |
(ftp)(http) |
TSV |
| GSM7030158_read_stats_WT_TC5_180_filtered.tsv.gz |
3.0 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
