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Sample GSM2837740

Query DataSets for GSM2837740

Status

Public on Nov 18, 2017

Title

mRNA_asf1Dset2D

Sample type

SRA

Source name

S.cerevisiae cells

Organism

Saccharomyces cerevisiae

Characteristics

genotype: asf1Dset2D
phase: mid-log phase
medium: YPD

Treatment protocol

For ChIP and MNase: Final 1% formaldehyde fixation and 125mM glycine quencing were performed before cell harvest.

Growth protocol

Cell culture were inoculated twice. Cell cultures were inoculated with shaking for aeration at proper temperatures.

Extracted molecule

total RNA

Extraction protocol

Every ChIP-seq cells were grown up to mid-log phase and harvested after final 1% formaldehyde fixation and 125mM glycine quencing. Cells were harvested by centrifugation at 4 Celcius degrees and the cell pellets were washed by 15mL of pH7.5 TBS buffer per a conical tube. ChIP cells were disrupted by glass bead vortexing in ChIP lysis buffer and sonicated by Sonic Dismembrator Model 500, Fisher Scientific. Debris of the sonicate was excluded by centrifugation by 14,800rpm at 4 Celcius degree. IP samples were aliqouted from the sonicate and immunoprecipiated by proper amount of antibody and protein A/G beads, washed by ChIP lysis buffer for over-night at 4 Celcius degrees. IP samples were washed and eluted by proper buffers in Sigmaprep spin column. Proteins in both IP and input samples were degraded by 2hr 30ug proteinase K rxn and de-crosslinked at 65 Celcius degrees for at least 8hrs. The IP and input DNA were eluted by Quiagen PCR purification kit.
Total RNA for mRNA-seq was prepared by the hot-phenol extraction.
For preparation of MNase-seq library, formaldehyde-fixed cell pellets were resuspended in 10 ml of pre-incubation buffer (20 mM EDTA, 0.7 M mercaptoethanol), washed, and resuspended in 2 ml of zymolyase buffer (1 M sorbitol, 50 mM Tris-Cl, pH 8.0, 5 mM mercaptoethanol) containing 2.5 mg ml-1 zymolyase (20T; US Biological). Lysed cells were spheroplasted at 30°C for 20 min and chromatin was isolated. Pelleted nuclei were digested with 200 units of MNase (NEB) at 37°C for 20 min (confirmed as a typical digestion condition by comparison of fragment sizes, data not shown), and then reverse cross-linked in 10 mM EDTA and 1% SDS buffer with proteinase K for 6 hours at 37°C. The DNA was extracted with phenol/chloroform, purified by ethanol precipitation, and resolved by 2% agarose gel electrophoresis in TAE buffer.
Yeast cells (2.5 million) were harvested at O.D. 0.6~0.8/mL (mid-log phase), and spheroplasted in 200 μl of Sorbitol buffer (1.4 M Sorbitol, 40 mM Tris-HCl, pH 7.5, 0.5 mM MgCl2) with 10 μl of 50 mg ml-1 of zymolyase (20T; US Biological). After washing twice with 100 μl of ice-cold Sorbitol buffer, the spheroplasts were incubated in fresh TD buffer (20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 20% dimethylformaldehyde) with transposase at 37°C for 4 hours. QIAquick PCR purification kit was used to purify DNA fragments, and transposition reactions and PCR amplifications were performed.
General Manual of Nextflex ChIP library preparation kit. mRNA isolation and mRNA-seq library preparation were performed using the NEXTflex Rapid Directional mRNA-Seq Bundle.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

The quality of fastq files were checked by fastqc and trimmed by trim_galore
The qualified fastq files were aligned to SacCer3 genome fa file using Bowtie2 v2.2.3. Then, the aligned files were compressed and sorted by samtools v1.3.
The sorted BAM files of biological duplicates were compared by MACS2, bam2wig and deeptools.
Genome_build: sacCer3
Supplementary_files_format_and_content: bigwig files were generated by MACS2, bam2wig and deeptools.

Submission date

Nov 02, 2017

Last update date

May 15, 2019

Contact name

Daeyoup Lee

E-mail(s)

[email protected]

Organization name

Korea Advanced Institute of Science and Technology

Department

Biological Sciences