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SRX10583013: GSM5238745: spt6-1004 Rpb1 ChIP-Seq replicate 1; Saccharomyces cerevisiae; ChIP-Seq
1 ILLUMINA (NextSeq 500) run: 11.1M spots, 463.3M bases, 209.6Mb downloads

Submitted by: NCBI (GEO)
Study: The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions
show Abstracthide Abstract
The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5' ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1. Overall design: ChIP-Seq of Abd1-HA, Spt5-V5, and Rpb1 in wild-type and spt6-1004 cells grown at 30oC and grown in triplicate
Sample: spt6-1004 Rpb1 ChIP-Seq replicate 1
SAMN18718135 • SRS8687152 • All experiments • All runs
Library:
Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: ChIP-seq was performed as described previously (Gopalakrishnan et al. 2019, Nucleic acids research) and is reproduced here. Cultures were cross-linked by the addition of formaldehyde to a final concentration of 1% followed by incubation with shaking at room temperature for 20 minutes. Glycine was added to a final concentration of 125 mM and the incubation was continued for 10 minutes. The cells were pelleted and washed twice with cold 1x TBS (100mM Tris, 150 mM NaCl, pH 7.5) and once with cold water. The cell pellets were then suspended in 800 µl cold LB140 buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1x cOMPLETE Protease Inhibitor tablet (Roche)). One ml of glass beads was added and the cells were lysed by bead beating for 8 minutes at 4oC with incubation on ice for 3 minutes after every one minute. The lysate was collected and centrifuged at 12,500 rpm for 5 minutes and the resulting pellet was washed once with 800 µl cold LB140 buffer. The pellet was resuspended in 580 µl cold LB140 buffer and sonicated in a QSonica Q800R machine for 20 minutes (30 seconds on, 30 seconds off, 70% amplitude). The sonicated samples were centrifuged at 12,500 rpm for 30 minutes and the resulting supernatant was taken for the immunoprecipitation step. 300-500 µg of S. cerevisiae chromatin was mixed with 33-55 µg (10%) of S. pombe chromatin and the volume was brought up to 800 µl with WB140 buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate). This was used as the input for the immunoprecipitation reaction. Antibody was added to the input and the samples incubated overnight at 4oC with end-over-end rotation. Fifty µl of Protein G sepharose beads (GE healthcare) pre-washed twice in WB140 was added to the IPs and samples were incubated for 4 hours at 4oC with end-over-end rotation. . The beads were washed twice with WB140, twice with WB500 (50 mM HEPES-KOH, pH 7.5, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), twice with WBLiCl (10 mM Tris pH 7.5, 250 mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate) for two minutes each and once with TE (10 mM Tris pH 7.4, 1 mM EDTA) for five minutes. The immunoprecipitated material was eluted twice with 100 µl TES (50 mM Tris pH 7.4, 1 mM EDTA, 1% SDS) at 65oC for 30 minutes. The eluates were incubated at 65oC overnight to reverse the crosslinking. Two hundred µl of TE was added to the eluates followed by RNase A/T1 to a final concentration of 0.02 µg/µl. The samples were incubated at 37oC for 2 hours. Proteinase K was added to a final concentration of 0.4 mg/ml and samples were incubated at 42oC for 2 hours. DNA was purified using Zymo DCC (for ChIP-Seq). The library preparation was done as described in Wong et al., 2013, Current protocols in molecular biology
Experiment attributes:
GEO Accession: GSM5238745
Links:
Runs: 1 run, 11.1M spots, 463.3M bases, 209.6Mb
Run# of Spots# of BasesSizePublished
SRR1421798911,131,375463.3M209.6Mb2021-09-12

ID:
14058885

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