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| Status |
Public on Oct 19, 2023 |
| Title |
L497-dhmU-MeDIP |
| Sample type |
SRA |
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| Source name |
L497-dhmU-MeDIP
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| Organism |
Breviolum minutum |
| Characteristics |
tissue: whole dinoflagellate
sample type: B. minutum 5-hmU MeDIP-seq
chip antibody: anti-5-hmU Abcam ab19735
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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 |
B. minutum genomic DNA was isolated as follows. Briefly, cells were centrifuged at 1,000 g for 5 minutes, then resuspended in 500 µL 1× Cell Lysis Buffer (prepared by mixing equal volumes of 2× Cell Lysis Buffer – 2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-HCl, pH 8.0 – and H2O) and vortexed. The lysed cells were mixed with an equal 500 µL volume Phenol:Chloroform:Isoamyl alcohol (25:24:1), and mixed well by inverting a few times. The phases were centrifugation at 13,000 g for 5 minutes, then the top phase was transferred to a new tube and treated with 4 µL Ribonuclease A (20 mg/mL) by incubating for 30 minutes at 37◦C. DNA was purified by adding an equal volume Phenol:Chloroform:Isoamyl alcohol (25:24:1), mixing well and centrifuging at 13,000 g for 5 minutes, then transferring the top layer to a new tube, to which Phenol:Chloroform:Isoamyl alcohol (25:24:1) was added again, and the centrifugation and top phase isolation was repeated. Then 2.5× volumes of 100% EtOH were added and the mixture was incubated on ice for 30 minutes or at -20◦C overnight. The solution was then centrifuged at 13,000 g at room temperature for 20 minutes, the pellet was washed with 70% EtOH, dried on air and resuspended in 50 µL H2O. To prepare inputs for MeDIP-seq experiments, gDNA was first sonicated using a Qsonica S-4000 with a 1/16” tip for 3 minutes, with 10 second pulses at intensity 3.5, and 20 seconds rest between pulses. For each reaction, 100 µL of Protein A Dynabeads (ThermoFisher Cat # 10002D) were washed 3 times with a 5 mg/mL BSA solution. Beads were then resuspended in 1 mL BSA solution and 5 µL of α-5-hmU antibody (Abcam Cat # ab19735) were added. Coupling of antibodies to beads was carried out overnight on a rotator at 4◦C. Beads were again washed 3 times with BSA solution and resuspended in 100 µL of BSA solution. Sheared genomic DNA (∼1 µg 1:1 mix of B. minutum and Homo sapiens) was end repaired and adapters were ligated to it following the procedure of the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S), purified using AMPure XP beads and eluted in 50 µL of H2O, and then denatured at 98◦C for 10 minutes. DNA was then immediately placed on ice, resuspended in 850 µL RIPA buffer (1× PBS, 1% IGEPAL, 0.5% Sodium Deoxycholate, 0.1% SDS, Roche Protease Inhibitor Cocktail) and added to the beads, then incubated overnight on a rotator at 4◦C. Beads were washed 5 times with LiCl buffer (10 mM Tris-HCl pH 7.5, 500 mM LiCl, 1% NP-40/IGEPAL, 0.5% Sodium Deoxycholate) by incubating for 10 minutes at 4◦C on a rotator, then rinsed once with 1× TE buffer. Beads were then resuspended in 200 µL IP Elution Buffer (1% SDS, 0.1 M NaHCO3) and incubated at 65◦C in a Thermomixer (Eppendorf) with interval mixing to dissociate antibodies. Beads were separated from the DNA solution by centrifugation, and DNA was purified using the MinElute kit.
Library generation was completed by carrying out PCR following the rest of the steps of the NEBNext Ultra II DNA Library Prep Kit protocol, using 15 cycles of amplification. Final libraries were purified using AMPure XP beads.
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
NextSeq 550 |
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| Description |
L497-dhmU-MeDIP
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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
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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 |
GPL33721 |
| Series (1) |
| GSE241969 |
Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome |
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| Relations |
| BioSample |
SAMN37205257 |
| SRA |
SRX21542001 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7746903_L497-dhmU-MeDIP.2x36mers.symbB.v1.0.unique.dedup.bigWig |
35.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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