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| Status |
Public on May 07, 2024 |
| Title |
MNase-ChIP, K36me2 ChIP, NSD RNAi S2 cells, Batch 5, Replicate 3 |
| Sample type |
SRA |
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| Source name |
S2
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2
cell type: Late-stage Embryonic
chip antibody: K36me2 (ab9049)
treatment: NSD RNAi
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| Treatment protocol |
RNAi against target genes in S2 cells was performed as previously described (Albig et al., 2016) with minor modifications. In brief, double-stranded RNA fragments (dsRNA) were generated with HiScribe T7 kit (NEB) from PCR products. Cells were incubated with 10 µg dsRNA per 1x10e6 cells (2 distinct sequences per target) in either 6 well plates or 75 cm^2 flasks for 1 hour. Two volumes of growth medium were added and cells were incubated at 26°C. Depending on the experiment, total duration of RNAi was 7-12 days with 2-3 rounds of fresh RNAi treatments.
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| Growth protocol |
Cells were cultured in Schneider’s Drosophila Medium (Thermo Fisher), supplemented with 10% heat-inactivated Fetal Bovine Serum (Sigma‑Aldrich), 100 units/mL penicillin and 0.1mg/mL streptomycin (Sigma‑Aldrich).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
70 to 130 million RNAi treated S2 cells, resuspended in 20 ml of complete Schneider’s Drosophila Medium, were crosslinked by adding 1:10 of the volume of Fixing Solution (100 mM NaCl, 50 mM Hepes pH 8, 1mM EDTA, 0.5 mM EGTA, 10% methanol-free formaldehyde) and rotated at room temperature for 8 min. 1.17 ml of freshly-prepared 2.5 M glycine was added to stop the fixation (final conc. 125 mM). Cells were pelleted at 500 g for 10 min (4°C) and resuspended in 10 mL of ice-cold PBS. 3.5 million of fixed D. virilis cells (fixed as described for D. melanogaster cells) were added for every 70 million D. melanogaster cells. Cells were pelleted at 526 g for 10 min (4°C) and resuspended in 1 ml of PBS + 0.5% Triton-X-100 + 1X cOmplete EDTA-free Protease Inhibitor for every 70 million D. melanogaster cells and rotated at 4°C for 15 min to release nuclei. Nuclei were pelleted at 2000 g for 10 min and washed once with 10 ml of ice-cold PBS. Nuclei were suspended in 1 ml of RIPA buffer (10 mM Tris-HCl pH 8, 140 mM NaCl, 1mM EDTA, 0.1% Na-deoxycholate, 1% Triton-X-100, 0.1% SDS, 1 mM PMSF, 1X cOmplete EDTA-free Protease Inhibitor + Roche 1x PhosSTOP) + 2 mM CaCl2 for every 70 million D. melanogaster cells, divided into 1 ml aliquots and flash-frozen in liquid N2. 1 mL of fixed nuclei was quickly thawed and 1 µL of MNase (to 0.6 units) (Sigma-Aldrich, Cat. No N5386) added. Chromatin was digested for 35 min at 37°C. MNase digestion was stopped by transferring the samples on ice and adding 22 µL of 0.5 M EGTA. Samples were mildly sonicated using a Covaris S220 instrument with the following settings: 50 W peak power, 20% duty factor, 200 cycles/burst, 8 min total time. Insoluble chromatin was removed by centrifugation at 16,000 g for 30 min at 4°C. Soluble chromatin was pre-cleared by incubation with 10 µL of 50% RIPA-equilibrated Protein A + G sepharose bead slurry (GE Healthcare, Cat. No 17-5280-11 and 17-0618-06) for every 100 µL of chromatin for 1 h at 4°C. 100 µL of pre-cleared chromatin were set aside (input) and kept overnight at 4°C, while each primary antibody was added to 300 µL of chromatin and incubated overnight at 4°C. 40 µL of 50% RIPA-equilibrated Protein A + G sepharose bead slurry was added for each immunoprecipitation and rotated 3 h at 4°C. Beads were washed 5 times with 1 ml of RIPA (5 min rotation at 4°C, pelleted at 3000 g for 1 min between washes) and resuspended in 100 µL of TE (10 mM Tris pH 8, 1 mM EDTA). 0.5 µL of RNaseA (Sigma-Aldrich, Cat. No. R4875) was added to both input samples and resuspended beads, followed by incubation at 37°C for 30 min. After addition of 6 µL of 10% SDS, protease digestion (250 ng/ µL Proteinase K, Genaxxon, Cat.no. M3036.0100) and crosslink reversal were performed simultaneously at 68°C for 2 hr. DNA was purified using 1.8X Agencourt AMPure XP beads (Beckman Coulter, Cat No A63880) following the standard protocol and eluted in 30 µL of 5 mM Tris-HCl pH 8. Libraries for sequencing were prepared using NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, E7465). Libraries were sequenced on an Illumina NextSeq 1000 instrument at the Laboratory of Functional Genomic Analysis (LAFUGA, Gene Center Munich, LMU).
Standard Paired-end protocol using 5-10ng input material using NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, E7465). Dual index oligos from NEB (#E6440). Libraries were size selected in 200-500 bp range and validated using Agilent TapeStation system.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 1000 |
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| Description |
DiffRegions_3kb_tol100_K36Me2_NSD_KD.bed
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| Data processing |
MNase-based paired-end ChIP-seq reads were demultiplexed using JEDemultiplexer and concordantly aligned using Bowtie2 with -X 500 and were filtered using samtools with parameter -q 10. Tag directories and coverage files (normalized to total number of virilis reads and to input) were generated by Homer Software package (Heinz et al. 2010).
Peak calling was performed on replicate-averaged normalized coverages using MACS2 v2.1.2 bdgpeakcall with parameters -l 1000 -c 3
Regions of differential K36me1/2/3 binding upon RNAi were identified using csaw v1.24.3 using BAM files obtained from bowtie2 for all replicates as input. 250bp windows of signficant difference were merged into regions of max 3kb.
Assembly: BDGP6 (dm6), release 104
Supplementary files format and content: For ChIPs, D. virilis scaled and input normalized .bigwig are provided for each individual sample. For inputs, representative D.virilis scaled .bigwig are provided.
Supplementary files format and content: Control K36me1/2/3 peaks as .bed (provided as supplementary to Control RNAi ChIP files)
Supplementary files format and content: csaw output of regions of significant change as .bed (provided as supplementary to HMT RNAi ChIP files except Batch0)
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| Submission date |
Jan 16, 2024 |
| Last update date |
May 07, 2024 |
| Contact name |
Muhunden Nallappa Jayakrishnan |
| E-mail(s) |
j_muhunden@hotmail.com, M.Jayakrishnan@bmc.med.lmu.de
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| Organization name |
Biomedical Center, Ludwig Maximilians University Munich
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| Department |
Molecular Biology
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| Lab |
Becker lab
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| Street address |
57 Nusselstrasse, 57
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| City |
Munich |
| State/province |
Munich (city) |
| ZIP/Postal code |
81245 |
| Country |
Germany |
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| Platform ID |
GPL33093 |
| Series (1) |
| GSE253391 |
Genomic context-dependent histone H3K36 methylation by Drosophila methyltransferases |
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| Relations |
| BioSample |
SAMN39462611 |
| SRA |
SRX23238299 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8019759_NSD_K36Me2_batch5_rep3.dvirNorm.dir.bw |
224.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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