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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 10, 2024 |
| Title |
Pol-I-Control-Rep1 |
| Sample type |
SRA |
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| Source name |
C64, Control, rep1
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| Organism |
Mus musculus |
| Characteristics |
cell line: C64
cell type: embryonic stem cell
antibody: Pol I, SCB
treatment: None
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| Treatment protocol |
mESCs were treated with 500nM auxin at 37 °C in a 5% CO2 incubator for 6 hours.
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| Growth protocol |
Cells were cultured on gelatin-coated plates in ESC media (Knockout DMEM, 15% FBS, 0.1mM MEM non essential amino acids, 2mM GlutaMAX, 0.1mM 2-mercaptoethanol and 1000 units/ml of ESGRO. ES cells are fed daily, cultured at 37C in a 5% CO2 incubator and passaged every two days via trypsinization.)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Extract protocol was done following the CUT&Tag published protocol where cells and DNA were sheared using 10uL 0.5M EDTA, 3uL 10% SDS, 2.5uL 20mg/mL Proteinase followed by phenol chloroform isoamyl extracted
Librarires were prepared following the CUT&Tag protocol. Briefly 21uL(out of 30uL) was used with 2uL Universal i5 primer (10uM) +2uL uniquely barcoded i7 primers (10uM) using a different unique barcode for each sample. 25UL of NEBNext HiFi 2x PCR Master mix (NEB M0541L) was used for the PCR reaction following the PCR protocol for CUT&Tag using 12 cycles. Library was cleaned up using Ampure XP beads using 1.1x volume and samples were pooled by volume and bead cleaned again before sequencing
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Quality Assurance was done using FastQC to check metrics
CUT&Tag reads were aligned to the mouse mm10 genome using bowtie2 with parameters: --no-unal –local –very-sensitive-local –no-discordant –no-mixed –contain –overlap –dovetail –phred33 -I 10 -X 9999 with PCR duplicates being kept
bigwig generation was done using the spike-in normalization factor or RPKM
Heatmaps, IGV tracks, and other downstream analysis were generated from bigwig files using Deeptools/ComputeMatrix
RPKM read counts were generated with DeepTools for Pol I, TBP and TRF2 using using bigwig files and the –outFileNameMatrix command with 10bp bins
ChrRNA-seq and NET-seq read counts were generated with bedtools multicov and each replicate was scaled to the spike-in scaling factor, summed and then log2 transformed
Replicates were merged using BigWigMerge
Assembly: mm10
Supplementary files format and content: bigWig, xlsx files with read counts
Library strategy: CUT&Tag
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| Submission date |
Jul 10, 2023 |
| Last update date |
Jul 10, 2024 |
| Contact name |
Sheila Teves |
| E-mail(s) |
TevesLab@gmail.com
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| Organization name |
Life Science Institute
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| Department |
Biochemistry
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| Lab |
Teves Lab
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| Street address |
2350 Health Sciences Mall
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| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V6T 1Ze |
| Country |
Canada |
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| Platform ID |
GPL30172 |
| Series (1) |
| GSE237001 |
Distinct modes of heat shock transcription factor interactions with mitotic chromosomes |
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| Relations |
| BioSample |
SAMN36397776 |
| SRA |
SRX20970425 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7592826_Pol-1-A-Ctrl-Rep1_S13.sorted.RPKM.bw |
10.3 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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