|
| Status |
Public on Jan 14, 2024 |
| Title |
anti-H3K4me3 - FLAG-Dsup - CUT&RUN |
| Sample type |
SRA |
| |
|
| Source name |
BY4741
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
genetic modification: Plasmid pRS306-PTDH3-Dsup
dsup strain: RGY002
antibody: EpiCypher # 13-0041 (Lot SG2419844A)
|
| Treatment protocol |
Ramazzottius varieornatus plasmid pRS306-PTDH3-Dsup (carrying N-terminally His tagged and C-terminally FLAG tagged Dsup) was digested with MfeI and integrated into the yeast strain BY4741 3 at the site of the endogenous TDH3 promoter to make the “Dsup” strain, RGY002. Further mutations of the Dsup gene, including deletion of the C-terminus to make the “Dsup ΔC” strain (RAY136), addition of an NLS to make “Dsup ΔC-NLS” (RAY228), substitution of R363E/R364E/R367E to make “Dsup 3R/3E” (RAY153), and addition of a stop codon at codon 2 of Dsup to make the “Empty vector” strain (RAY149), were made after integration using CRISPR-Cas9 mediated genome editing. See referenced text for further detail.
|
| Growth protocol |
Yeast were grown in 500 mL of SC-ura media to OD of 0.6-0.8. Cells were spheroplasted using 500 μL of 2 mg/mL Zymolyase 100T at 37°C for approximately 30 mins (until a 50 μL aliquot mixed with 1 mL of 10% SDS had an OD approximately 10% of the starting value). See referenced text for further detail.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei from yeast cells expressing Dsup or mutant Dsup were purified according to published methods (doi:10.1007/978-1-0716-2257-5_9) with slight modifications described above. 1 mL aliquots containing approximately 5 x 107 nuclei were slow-frozen in an isopropanol chamber at -80°C overnight. Immediately prior to an experiment, nuclei were thawed rapidly for 2-3 minutes at 37°C, then 100 μL of nuclei suspension was used per CUT&RUN reaction with the CUTANA™ ChIC/CUT&RUN Kit version 3 (EpiCypher). The protocol was carried out as directed by the version 3.2 user manual. MNase digestion of chromatin was performed for 2 hours at 4°C. See referenced text for further detail.
5 ng of DNA was used to prepare sequencing libraries with the Ultra II DNA Library Prep Kit (NEB #E7645L). Libraries were sequenced on an Illumina NextSeq 2000 platform. See referenced text for further detail.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
Dsup_H3K4me3
|
| Data processing |
*library strategy: CUT&RUN
Paired-end fastq files were aligned to thesacCer3reference genome using Bowtie2 (v2.2.5).
Duplicate reads were removed via SAMtools (v1.6).
Multi-aligned reads were removed via Picard (v2.26.2).
bigWig creation included both E. coli normalization and RPKM normalization via bamCoverage in deeptools (v3.5.1). Files were E. coli normalized by an E. coli scaling factor (1/%E. coli reads) and RPKM normalized ( --normalizeUsing option).
Assembly: sacCer3
Supplementary files format and content: bigWig
|
| |
|
| Submission date |
Jul 14, 2023 |
| Last update date |
Jan 14, 2024 |
| Contact name |
Michael-Christopher Keogh |
| E-mail(s) |
mkeogh@epicypher.com
|
| Organization name |
EpiCypher, Inc
|
| Street address |
6 Davis Dr
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27560 |
| Country |
USA |
| |
|
| Platform ID |
GPL31112 |
| Series (1) |
| GSE237436 |
Multivalent binding of the tardigrade Dsup protein to heterologous chromatin promotes yeast survival and longevity upon exposure to oxidative damage |
|
| Relations |
| BioSample |
SAMN36458069 |
| SRA |
SRX21026407 |
