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| Status |
Public on Mar 27, 2024 |
| Title |
WCE_FBY2732_2 |
| Sample type |
SRA |
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| Source name |
S. pombe cells
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: S2A Set2-TAP
fraction: input
spike-in: none
genotype: h- rpb1 S2A-kanR set2-TAP-hphR
cell type: S. pombe cells
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| Treatment protocol |
Cultures were cross-linked with 1% formaldehyde for 20 min at room temperature with manual shaking every 5 min.
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| Growth protocol |
For each condition (IP), 50 mL of cell cultures were grown to an OD600nm of ∼0.5 at 30 °C in YES .
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After quenching the solution with glycine (final concentration of 360 mM), cells were washed twice with a cold Tris-buffer saline (20 mM Tris-HCl [pH 7.5] and 150 mM NaCl) and frozen in liquid nitrogen. Cell pellets from 50 ml cultures were thawed and resuspended in 500 µL of cold lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100 and 0.1% Na-deoxycholate) that was supplemented with a protease inhibitor cocktail (1X PMSF and 1X PLAAC) and disrupted vigorously with glass beads in a FastPrep-24TM (MP biomedical) for 3 cycles of 30 sec at 6.5 m/s. Then, 150 µL of cold lysis buffer with proteases was added to increase the volume and samples were sonicated for 12 cycles of 10 sec at 20% amplitude with a Branson digital sonifier. Sonicated chromatin was then incubated overnight at 4 °C with 50 µl of magnetic beads IgG Dynabeads (Life Technologies, 11041) in the case of TAP tagged proteins, or Dynabeads M-280 Sheep Anti-Rabbit IgG (Invitrogen, 11203D) beads coupled with 2 ug of rabbit anti-Histone H3 total (ab1791, abcam), H3K36me3 (ab9050, abcam) or H3K9ac (06-942, Millipore Sigma). Beads were then washed twice for 4 minutes at 4oC with 1 mL of cold lysis buffer, twice with 1 mL of cold lysis buffer with 500 mM NaCl, twice with 1 mL of cold wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate and 1 mM EDTA), and once with 1 mL of cold Tris-EDTA (TE: 10 mM Tris-HCl [pH 8.0] and 1 mM EDTA [pH8.0]). Bound material was eluted by resuspending the beads in 50 µl elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA and 1% SDS) at 65 °C for 15 min at 1200 rpm in an Eppendorf Thermomixer. After a short centrifugation, reverse-crosslinking was performed by incubating eluted chromatin with 120 µl of TE and 1% SDS or 5 µl of WCE in 165 µl of TE with 1% SDS at 65 °C overnight. For ChIP-Seq experiments, reverse-crosslinking was perfomed by incubating eluted chromatin with 20 µl of WCE in 150 µl of TE and 1% SDS. Samples were treated with a proteinase K mix (150 mg proteinase K, 60 mg glycogen in TE) and DNA was extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1, [pH8.0], Invitrogen) and once with chloroform (Bioshop). Following ethanol precipitation, pellets were resuspended in 30 µl of TE plus 10 µg of RNAse A and incubated 1 h i at 37 °C. RNase-treated DNA was then purified with a QIAGEN PCR purification kit. The input DNA was diluted 400 times while the coimmunoprecipitated DNA was diluted 100 times in water. DNA were analyzed on a LightCycler 96 Instrument system (Roche) with the addition of the 2X SYBR Green in the presence of 0.15 μM of gene-specific oligonucleotide
ChIP-sequencing libraries were prepared using the SPARQ DNA Sample Prep Kit Illumina Platform (Quantabio) according to the manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Reads were mapped using HISAT2 (Kim et al, 2015) with option --no-splice-alignment on a concatenate genome containing the S. pombe (version ASM294v2.26) and S. cerevisiae chromosomes. Normalization factors were computed based on the proportion of reads mapped to the S. cerevisiae genome as previously described (Larochelle et al, 2018) while the average fragment size was estimated using MACS2 predicted function (Zhang et al, 2008). Based on the normalization factors and averaged fragment size, we used deepTools (Ramirez et al, 2016) to generate normalized coverage files (.bigwig) with options bamCoverage --smoothlength 20 --centerReads -e [average_fragment_size] --scaleFactor [size_factor] -bs 1 -of bigwig. If no spike-in was available (as it is the case for the Pcf11-myc and Set2-TAP ChIP data), the option --normalizeUsing RPKM was used instead of --scale factor.
Assembly: ASM294v2.26
Supplementary files format and content: genome coverage files in bigWig format are provided
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| Submission date |
Dec 03, 2023 |
| Last update date |
Mar 27, 2024 |
| Contact name |
Carlo Yague-Sanz |
| E-mail(s) |
yague.carlo@gmail.com
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| Organization name |
Namur University
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| Department |
Medecine
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| Lab |
GEMO lab
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| Street address |
61 rue de Bruxelles
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| City |
Namur |
| ZIP/Postal code |
5000 |
| Country |
Belgium |
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| Platform ID |
GPL20584 |
| Series (2) |
| GSE249234 |
Proximity-dependent biotinylation map of the RNAPII CTD reveals that the primary role of Serine 2 phosphorylation is in the suppression of pervasive antisense transcription and not in RNA 3’ end processing [ChIP-seq] |
| GSE249236 |
Proximity-dependent biotinylation map of the RNAPII CTD reveals that the primary role of Serine 2 phosphorylation is in the suppression of pervasive antisense transcription and not in RNA 3’ end processing |
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| Relations |
| BioSample |
SAMN38606519 |
| SRA |
SRX22722550 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7928788_9_FBY2732_CTD-S2A_Set2-TAP.bam.bw |
80.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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