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| Status |
Public on Feb 01, 2024 |
| Title |
02-02_Epe1-Flag_WT |
| Sample type |
SRA |
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| Source name |
yeast cells
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
tissue: yeast cells
genotype: Epe1-Flag-WT
strain: HSK522
description: chip_antibody: anti-Flag (Sigma F1804)
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| Growth protocol |
All yeast cells were grown in rich medium (YES)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin Immunoprecipitation (ChIP) experiments were performed as previously described29. Briefly, freshly grown 40 ml cells in YES were fixed with 1% formaldehyde from 37% stock (Sigma F8775) for 20 min at room temperature for H3K9me2, H3K9me3, RNA Pol II, Ago1 and Chp1 ChIP. For Swi6 and 3xFlag-Clr4 ChIP, 40 ml cells were pre-incubated at 18 °C for 2 h and fixed with 1.45% Formaldehyde from 16% stock (ThermoFisher 28908) for 30 min at 18 °C. Cells were quenched with 360 mM glycine and 2.4 mM Tris for 5 min. Whole-cell extracts (WCE) were prepared using FastPrep-24 and sonicated using Covaris. 1~2 μg of antibodies like anti-H3K9me2 (Abcam ab1220), anti-H3K9me3 (Absolute Antibody Ab00700-1.26), anti-RNA polymerase II (BioLegend 904001), Swi6 (Abcam ab188276), Ago1 (Abcam Ab18190), Chp1 (Abcam ab18191) and anti-Flag (Sigma F1804) were pre-incubated with 50 μl Protein A and Protein G sepharose (GE 17513801 and 17061801) mix for 3~4 h and then incubated with sheared chromatin overnight at 4 °C. After washing, bead-antibody-chromatin mixture were treated with Proteinase K (ThermoFisher 100005393) for 1 h ant 42 °C and incubated 5~6 h at 65 °C to reverse cross-links. DNA was cleaned up using ChIP DNA Clean & Concentrator (Zymo Research D5205) and subsequently used for qPCR or ChIP-Seq. For sRNA-seq, Total RNAs were purified from freshly grown yeast cells using Quick-RNA Fungal/Bacterial Miniprep Kit (Zymoresearch R2014) and sRNA were enriched using RNA Clean & Concentrator (Zymoresearch R1013).
ChIP-Seq libraries were constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645) and barcoded multiplex libraries were sequenced using Illumina MiSeq platform, which generates paired-end 101 bp reads. sRNA libraries were constructed using NEBNext Small RNA Library Prep Set for Illumina (NEB E7330) and barcoded multiplex libraries were sequenced using Illumina MiSeq platform, which generates single-end 36 nt reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Data processing |
ChIP-seq reads were trimmed using Trimmomatic, mapped to S. pombe genome using Bowtie2. Bigwig files were generated using bamCoverage and visualized in IGV genome browser. Total ChIP-seq read counts for defined region were analyzed using MultiBigwigSummary or MultiBamSummary.
sRNA-seq reads were adapter-trimmed using FASTX-Toolkit, keeping a minimum read size of 15 nt, and mapped to the S. pombe genome using Bowtie allowing up to 1 mismatch. The strand-specific genomic coverage was calculated using Bedtools41 and represented in 20 nt windows.
Assembly: ASM294v2
Supplementary files format and content: Bigwig
Supplementary files format and content: Bedgraph
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| Submission date |
Jul 27, 2022 |
| Last update date |
Feb 01, 2024 |
| Contact name |
Hyun Soo Kim |
| E-mail(s) |
hskim@cshl.edu
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| Organization name |
Cold Spring Harbor Laboratory
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| Department |
Plant
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| Lab |
Martienssen
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| Street address |
1 Bungtown Rd
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| City |
Cold Spring Harbor |
| State/province |
New York |
| ZIP/Postal code |
11724 |
| Country |
USA |
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| Platform ID |
GPL16192 |
| Series (1) |
| GSE156069 |
Clr4SUV39H1 ubiquitination and non-coding RNA mediate transcriptional silencing via heterochromatic phase transitions |
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| Relations |
| BioSample |
SAMN30004590 |
| SRA |
SRX16700197 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6409937_02-02_Epe1-Flag_WT.bigwig |
1.3 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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