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| Status |
Public on Jun 15, 2016 |
| Title |
jhd2Δ H3K4me2 repeat1 |
| Sample type |
SRA |
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| Source name |
sonicated soluble chromatin
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| Organisms |
Saccharomyces cerevisiae; Nakaseomyces glabratus |
| Characteristics |
strain: YMC70
genotype: jhdelta2_delta::KanMX Mat a hta1-htb1delta hta2-htb2delta lys2-128deltahis3delta_200 ura3-52 HTA1-HTB1 (CEN HIS3)
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| Growth protocol |
Yeast were grown in YPAD broth (1% Yeast extract, 2% Peptone, 2% Dextrose, and 0.004% adenine hemisulfate) at 30°C with constant shaking at 225 rpm to an OD A600 of 0.8-1.0.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Yeast strains were grown to mid-log phase and cross-linked with 1% formaldehyde for 20 min at room temperature. Crosslinking was stopped using 0.125 mM glycine and cells were washed with 1X PBS. Multiple aliquots of 40 × 107 cross-linked cells each were prepared. Candida glabrata (Anderson) Meyer et Yarrow Strain Designation: NCYC 388 (ATCC® 36909) was grown at 25°C and subjected to formaldehyde crosslinking as described for budding yeast, and individual aliquots containing 20 × 107 cells each were harvested. Cross-linked S. cerevisiae (40 × 107) and C. glabrata (20 × 107) cells were mixed together in FA140+SDS buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mM NaCl, 0.1% SDS) and lysed by bead beating. Lysates from two sets of S. cerevisiae and C. glabrata admixture (i.e., 120 × 107 cells in total) were pooled and sonicated in a Diagenode Bioruptor™ for 30 min (6 cycles of 5 min each, 30 sec ON and 30 sec OFF at high setting). Soluble chromatin was obtained after two sequential high-speed centrifugations of the sonicated lysate (16,100 X g for 5 min and 15 min). SDS was removed by dialysis as follows. Soluble chromatin obtained from four batches of control or mutant budding yeast and candida admixture were pooled, transferred to a 5 ml Float-A-Lyzer G2 dialysis device (3.5 - 5 kD MWCO) (Spectra/Por) and dialyzed initially for 2 h in 1 L FA140 buffer followed by an overnight dialysis in fresh 4 L FA140 buffer. Dialysate was centrifuged at 16,100 X g for 15 min at 4°C. A 100 μl aliquot was set aside for isolating input DNA. The remaining dialysate was split equally into three tubes and pre-cleared using 50 μl blocked Dynabeads® Protein A for 1h at 4°C with end-over-end rotation. Dynabeads® Protein A were blocked with BSA and fish skin gelatin as described above except bead blocking was done for 4 h. Pre-cleared lysate was combined with either α-H3K4me1 (20 μl, Active Motif) or α-H3K4me2 (10 μl, Millipore) or α-H3K4me3 (10 μl, Active Motif) antibody and 100 μl blocked Dynabeads® Protein A, and incubated overnight at 4°C with end-over-end rotation. Beads were initially washed with three times with FA140 buffer by inversion followed by sequential washes (5 min each with end-over-end rotation): FA140 buffer (2 times), FA1000 buffer (2 times), FA500 (2 times), LiCl/NP40 buffer (1 time) and TE (1 time). ChIP DNA was eluted in 400 μl elution solution (1% SDS, 100 mM sodium bicarbonate) and incubated at room temperature with end-over-end mixing for 40 min. The eluate is transferred to a fresh 0.65 ml tube, 18 μl 5 M NaCl is added and mixed well. At this stage, 400 μl elution solution and 18 μl 5 M NaCl is also added to the 50 μl soluble chromatin set aside to isolate ‘‘input’’ DNA. Reverse crosslinking is done by incubation at 65 °C for 5 h in a thermal cycler. The solution was transferred to a fresh 1.5 ml tube, 1 ml ethanol was added, mixed well and precipitation was allowed to occur overnight at -20 °C. The samples were centrifuged at maximum speed in a table-top centrifuge at 4°C. The pellet was washed with 70% ethanol followed by centrifugation at high speed. The pellet was dissolved in 90 μl water, treated with 2 μl 10 mg/ml RNase A and followed by an incubation at 37 °C for 30 min. Proteins were removed by adding 10 μl 10X PNK buffer (100 mM Tris.Cl, pH 8.0, 50 mM EDTA, 5% SDS), 1 μl Proteinase K (Sigma) and incubating at 42°C for 1 h. DNA was purified using Qiaquick PCR Purification kit after addition of 20 μl 3 M Sodium acetate to adjust the pH. DNA was eluted in 50 μl water and stored at -20 °C. DNA concentration was measured using Qubit Fluorometer. Equal amount ChIP or input DNA was used in library construction using NEBNext® Multiplex Oligos (Index Primers Set 1 and Index Primer Set 2) and NEBNext® ChIP-seq Library Prep Reagent Set for Illumina.
Equal amount of purified DNA (10ng) was subjected to library construction using the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina following the manufacturer's protocol. Adaptor-ligated and index primer-containing amplified DNA were resolved in an 2.0% agarose gel and 300-500 bp sized fragments were purified using the Qiagen Minelute Gel Extraction Kit, except incubation to melt the gel was performed at 37oC for 30min.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ChIP DNA
contains DNA from Candida glabrata (Anderson) Meyer et Yarrow Strain Designation: NCYC 388 (ATCC® 36909) used as calibration genome
calibrated processed data file: Jhd2-K4ME2.calibrated.bw
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| Data processing |
De-multiplexed Fastq files from the Illumina pipeline were aligned using Novoalign version 2.8 (http://www.novocraft.com), allowing for one random repeat, base recalibration, and a minimum alignment quality score of 13. Sequences were aligned first to the C glabrata genome; unaligned read sequences were exported and re-aligned to the S cerevisiae genome. To identify orthologous alignments, aligned sequences to the C glabrata genome were exported as fastq and re-aligned to S cerevisae; sequences that aligned to both genomes were considered orthologous. Alignments were converted to sorted, indexed Bam files using samtools. Biological replicates, after comparison, had their alignments were merged using samtools.
Fragment coverage was generated using the BioToolBox bam2wig application (v1.35) by extending each alignment in the 3' direction by 200 bp and counting the coverage. Depth was normalized to reads per million (RPM). Coverage tracks were converted to bigWig files for visualization and analysis using UCSC utilities.
Replicates were compared by generating a Pearson correlation of the coverage files using the UCSC utility bigWigCorrelate with a max threshold of 250. Replicates were merged with samtools and coverage tracks re-generated.
The number of alignments to S cerevisiae and C glabrata were counted using samtools. The number of orthologous alignments were subtracted from C glabrata counts. A normalization factor was derived from the formula (Cg_Ref x Sc_ChIP) / (Sc_Ref x Cg_ChIP), where Cg_Ref is the number of Input alignments to C glabrata, Sc_ChIP is the number of ChIP alignments to S cerevisiae, Sc_Ref is the number of Input alignments to S cerevisiae, and Cg_ChIP is the number ChIP alignments to C glabrata. This normalization scale was then applied to every genomic position represented in a bedGraph file of the ChIP fragment coverage to S cerevisiae using the script manipulate_wig (https://github.com/tjparnell/HCI-Scripts).
Genome_build: SacCer3
Genome_build: Candida glabrata, GCA_000002545.2, Ensembl Fungi release 30
Supplementary_files_format_and_content: BigWig files representing uncalibrated fragment (uniformly extended to 200 bp) coverage depth normalized to reads per million mapped. Filenames have uncalibrated in their name.
Supplementary_files_format_and_content: BigWig files representing the the merged replicate, calibrated fragment coverage depth. Filenames have calibrated in there name. These files are available in a tar archive on the series record.
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| Submission date |
May 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Timothy J Parnell |
| E-mail(s) |
timothy.parnell@hci.utah.edu
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| Organization name |
Huntsman Cancer Institute
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| Street address |
2000 Circle of Hope
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| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
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| Platform ID |
GPL21803 |
| Series (2) |
| GSE73407 |
Set1 H3K4 methyltransferase and Jhd2 H3K4 demethylase |
| GSE81020 |
Regulation of chromatin structure by Set1 H3K4 methyltransferase and Jhd2 H3K4 demethylase [H3K4me ChIP-seq] |
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| Relations |
| BioSample |
SAMN04932278 |
| SRA |
SRX1738618 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2140742_Jhd2-K4ME2_rep1.uncalibrated.ext200.rpm.bw |
19.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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