 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 21, 2022 |
| Title |
myoLuc_0h_H3K27ac_R2 |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic Fibroblasts
|
| Organism |
Myotis lucifugus |
| Characteristics |
cell type: embryonic fibroblast
antibody: Rabbit anti-H3K27ac EMD (Millipore #MABE647)
treatment: untreated
timepoint: untreated
|
| Treatment protocol |
For CUT&RUN, cells were seeded in 15cm plates (2,000,000 cells per dish). After 48hrs culturing media was replaced with fresh media containing either 1000U/mL universal IFNA (PBL Assay Science #11200) or equal volume of DPBS. Cells were treated for 4hrs.
|
| Growth protocol |
Myotis lucifugus embryonic fibroblast cells were routinely grown at 37C in 5% CO2 on coated plastic (VWR #10861-680, typically 1,000,000-2,000,000 cells in 10cm dishes) in DMEM (cat #10566016, ThermoFisher) supplemented with 10% FBS, 5% MEM nonessential amino acids, 100 U/mL penicillin-streptomycin, and 1 mM sodium pyruvate.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&RUN pulldowns were generated using a protocol from Meers et al. 2019 (PMID: 31232687, https://dx.doi.org/10.17504/protocols.io.zcpf2vn). All buffers were prepared according to the “High Ca2+/Low Salt” section of the protocol using 0.04% digitonin. 500,000 viable cells were used for each pulldown. The following antibodies were used: rabbit anti-mouse IgG (1:100, Abcam #ab46540), rabbit anti-H3K27ac (1:100, Millipore #MABE647), rabbit anti-pPOLR2A-Ser5 (Cell Signaling #13523S), rabbit anti-STAT1 (1:100, Cohesion Biosciences #3322), rabbit anti-pSTAT1-Ser727 (1:100, Active Motif #39634). pAG-MNase was added to each sample following primary antibody incubation at a final concentration of 700 ng/mL. Chromatin digestion, release, and extraction was carried out according to Meers et al. 2019 (PMID: 31232687, https://dx.doi.org/10.17504/protocols.io.zcpf2vn). Pulldown success was determined by Qubit dsDNA High Sensitivity (Life Technologies #Q32851) and TapeStation 4200 HSD5000 (Agilent #50675588) before proceeding with library preparation.
Libraries were generated using a modified protocol for use with the KAPA HyperPrep Kit (Roche #KK8502). Briefly, the full volume of each pulldown (~30 uL) was diluted to 50 uL in nuclease-free water, and libraries were generated following the manufacturer’s protocol with the following modifications. Freshly diluted 0.200 uM single-index adapters were added to each library at a low concentration (9 nM) to minimize adapter dimer formation. Adapter-ligated libraries underwent a double-sided 1.0X/1.2X cleanup using KAPA Pure Beads (Roche #07983280001). Purified, adapter-ligated libraries were amplified using the following PCR cycling conditions: 45 s at 98°C, 15x(15 s at 98°C, 10 s at 60°C), 60 s at 72°C. Amplified libraries underwent a double-sided 1.1/1.2X cleanup. The final libraries were quantified using Qubit dsDNA High Sensitivity and TapeStation 4200 HSD5000. Libraries were pooled and sequenced on an Illumina NovaSeq 6000 (Novogene) as 150bp paired-end reads.
CUT&RUN
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Real-Time Analysis v3.4.4 was used for basecalling.
Adapters and low quality reads were trimmed using BBDuk v38.05 using options ‘ktrim=r k=34 mink=11 hdist=1 tpe tbo qtrim=r trimq=10’. Trimmed reads were aligned to the myoLuc2_HiC assembly using BWA-MEM v0.7.15, and only uniquely mapping reads with a minimum MAPQ of 10 were retained. Fragments aligning to the mitochondrial genome (HiC_scaffold_93) were removed. Peak calling was performed using alignment files with MACS2 v2.1.1 in a two-step process where separate sets of peaks were called with 1) single-end options ‘--format BAM --shift=-75 --extsize=150’ and 2) paired-end option ‘--format BAMPE’. For both modes only peaks with a p-value < 0.01 were retained. Independently, peaks were called from IgG control libraries using MACS2 v2.1.1 with paired-end option ‘--format BAMPE’ and subtracted from pulldown peak files. Bigwig files were prepared from the MACS2 normalized bedgraph files using bedGraphToBigWig v4.
Assembly: myoLuc2_HiC
Supplementary files format and content: Normalized signal tracks corresponding to read coverage per 1 million fragments in UCSC bigwig format
|
| |
|
| Submission date |
Apr 14, 2022 |
| Last update date |
Apr 23, 2022 |
| Contact name |
Edward B Chuong |
| E-mail(s) |
edward.chuong@colorado.edu
|
| Organization name |
University of Colorado Boulder
|
| Department |
Molecular, Cellular and Developmental Biology
|
| Lab |
Chuong Lab
|
| Street address |
3415 Colorado Ave., room E251
|
| City |
Boulder |
| State/province |
Colorado |
| ZIP/Postal code |
80303 |
| Country |
USA |
| |
|
| Platform ID |
GPL32169 |
| Series (2) |
| GSE200831 |
Transcriptional dynamics of transposable elements in the type I IFN response in Myotis lucifugus cells (CUT&RUN) |
| GSE200833 |
Transcriptomic and epigenomic profiling of Myotis lucifugus embryonic fibroblast cells |
|
| Relations |
| BioSample |
SAMN27589864 |
| SRA |
SRX14860888 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6044777_myoLuc_0h_H3K27ac_R2.bw |
31.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
