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| Status |
Public on Oct 19, 2023 |
| Title |
L1634-DVNP_GM05_dSMF_EM-seq |
| Sample type |
SRA |
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| Source name |
L1634-DVNP_GM05_dSMF_EM-seq
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| Organisms |
Saccharomyces cerevisiae; Candida albicans |
| Characteristics |
tissue: whole yeast
sample type: yeast expressing B. minutum DVNP symbB.v1.2.006931 SMF
chip antibody: n/a
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| Growth protocol |
(B. minutum) The clonal axenic Symbiodinium/Breviolum minutum strain SSB01 was used. Stock cultures were grown in Daigo's IMK medium for marine microalgae (Wako Pure Chemicals) supplemented with casein hydrolysate (IMK+Cas) at 27C at a light intensity of 10 µmol photons m-2s-1 from Philips ALTO II 25-W bulbs on a 12-h-light:12-h-dark cycle. The medium was prepared in artificial seawater (ASW).
(yeast) Standard yeast cell culture practices were followed. Cells were grown in YPD media at 30C until harvesting.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Yeast SMF experiments were carried out as follows. A 1:1 mixture of S. cerevisiae cells expression DVNPs and Candida albicans cells (used as a control for normalization) amounting to a total of 2.5×108 cells was used as input. Cells in log phase (OD660 ≤ 1.0) were first centrifuged at 13,000 rpm for 1 minute, then washed with 100 µL Sorbitol Buffer(1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2), and centrifuged again at 13,000 rpm for 1 minute. Cells were then spheroplasted by resuspending in 200 µL Sorbitol Buffer with DTT added at a final concentration of 10 mM and 0.5 mg/mL 100T Zymolase, followed by incubating for 5 minutes at 30◦C at 300 rpm in a Thermomixer. The pellet was centrifuged for 2 minutes at 5,000 rpm, washed in 100 µL Sorbitol Buffer, and centrifuged again at 5,000 rpm for 2 minutes. Cells were then resuspended in 100 µL ice-cold Nuclei Lysis Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, 0.5% NP-40) and incubated on ice for 10 minutes. Nuclei were then centrifuged at 5000 rpm for 5 min at 4◦C, resuspended in 100 µL cold Nuclei Wash Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA), and centrifuged again at 5,000 rpm for 5 min at 4◦C. Finally, nuclei were resuspended in 100 µL M.CviPI Reaction Buffer (50 mM Tris-HCl pH 8.5, 50 mM NaCl, 10 mM DTT). Nuclei were then first treated with M.CviPI (GpC methyltransferase) by adding 200 U of M.CviPI (NEB), SAM at 0.6 mM and sucrose at 300 mM, and incubating at 30◦C for 7.5 min. After this incubation, 128 pmol SAM and another 100 U of enzymes were added, and a further incubation at 30◦C for 7.5 min was carried out. Immediately after, M.SssI treatment (CpG methyltransferase) followed, by adding 60 U of M.SssI (NEB), 128 pmol SAM, MgCl2 at 10 mM and incubation at 30◦C for 7.5 min. The reaction was stopped by adding an equal volume of Stop Buffer (20 mM Tris-HCl pH 8.5, 600 mM NaCl, 1% SDS, 10 mM EDTA). HMW DNA was isolated using the MagAttract HMW DNA Kit (Qiagen; cat # 67563) following the manufacturer’s instructions.
Enzymatically labeled DNA was then sheared on a Covaris E220 and converted into sequencing libraries following the EM-seq protocol, using the NEBNext Enzymatic Methyl-seq Kit (NEB, Cat # E7120L).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
L1634-DVNP_GM05_dSMF_EM-seq
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| Data processing |
(read alignment, ATAC-seq and MeDIP-seq data) Bowtie 1.0.1
(bigWig file generation) custom code
(read alignment, SMF data) bwa-meth
(read alignment, 5-hmU chemical conversion data) slamdunk
Assembly: custom and GCA_000507305.1_ASM50730v1 for Breviolum minutum, hg38 for human, sacCer3 for S. cerevisiae; C_glabrata_CBS138 for Candida albicans
Supplementary files format and content: bedGraph of read coverage, normalized to RPM for bigWig files for ATAC-seq and MeDIP-seq; slamdunk output for 5-hmU conversion datasets; MethylDacket bedGraph output for SMF datasets
Library strategy: SMF
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| Submission date |
Aug 30, 2023 |
| Last update date |
Oct 19, 2023 |
| Contact name |
Georgi Kolev Marinov |
| Organization name |
STANFORD UNIVERSITY
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| Department |
Genetics
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| Street address |
279 Campus Drive West, Beckman Center, B-257A/259
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| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305-5101 |
| Country |
USA |
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| Platform ID |
GPL33723 |
| Series (1) |
| GSE241969 |
Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome |
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| Relations |
| BioSample |
SAMN37205248 |
| SRA |
SRX21542012 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7746912_L1634-DVNP_GM05_dSMF_EM-seq.bwameth.sacCer3+C_glabrata_CBS138_current_chromosomes.dedup_CHH.bedGraph.gz |
10.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
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