 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 16, 2022 |
| Title |
Pou2f3.mTEC.rep#7 |
| Sample type |
SRA |
| |
|
| Source name |
Thymus
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: WT
cell type: mTEC
|
| Treatment protocol |
mTEC were prepared by digesting thymi with collagenase and dispase, magnetically depleting CD45+ cells, staining with fluorescent antibodies, and flow sorting mTECs into DMEM/10%FCS.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
mTECs were washed in wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine), bound to concavalin A beads (Bangs Laboratories), permeabilized in wash buffer plus 0.05% digitonin, and incubated overnight with 1:50 primary antibody (anti-H3K27ac [Abcam], -Hnf4α [Abcam], -Grhl1 [Novus], -Pou2f3 [Sigma], or -IgG [Rockland]) at 4°C in wash buffer plus 0.05% digitonin, 2mM EDTA and 0.1% BSA. The next day, H3K27ac, Grhl1, IgG, and some Pou2f3 samples were lightly fixed with 0.1% paraformaldehyde at room temperature for 2 minutes before proceeding. (We and others have observed improved CUT&Tag signal-to-noise ratio for some TFs with light fixation.) Samples were incubated with secondary antibody (guinea pig anti-rabbit [Rockland] for H3K27ac, Grhl1, Pou2f3, and IgG; rabbit anti-mouse [Abcam] for Hnf4α) at 1:100 for 1 hour at room temperature in wash buffer plus 0.05% digitonin. Samples were washed and incubated with pA-Tn5 (Addgene #124601, gift from Steve Henikoff, purified in-house) at 1:200 for 1 hour at room temperature in 300mM NaCl wash buffer plus 0.01% digitonin. Samples were washed twice then tagmented for 1 hour at 37°C in 300mM NaCl wash buffer plus 0.01% digitonin and 10mM MgCl.
Tagmentation was halted with EDTA, sodium dodecyl sulfate and proteinase K for 1 hour at 55°C. Tagmented DNA was phenol-chloroform extracted and amplified by PCR with NEBNext 2X Master Mix (NEB) using the following program: 72°C for 2min, 98°C for 30s, 16 cycles of 98°C for 10s and 63°C for 10s, 72°C for 1min, hold. Amplified libraries were quantified by Qubit (Thermo) and Tapestation (Agilent).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
CUT&Tag
|
| Data processing |
Library strategy: CUT&Tag
Low quality reads were discared using Trimmomatic (v0.36)
Reads were mapped to the mouse reference genome (mm10) with bowtie2 (v2.29) using parameters --local --very-sensitive -no-mixed --no-discordant -I 10 -X 700
Bam files were generated and multimapping reads removed by samtools (v1.3.1), and duplicate reads were removed by picard (v2.8.0)
Bigwig files were generated using deeptools (v3.0.2) with counts-per-million normalization
Genome_build: mm10
Supplementary_files_format_and_content: bigWig
|
| |
|
| Submission date |
Jan 23, 2022 |
| Last update date |
Jun 16, 2022 |
| Contact name |
CBDM Lab |
| E-mail(s) |
cbdm@hms.harvard.edu
|
| Phone |
617-432-7747
|
| Organization name |
Harvard Medical School
|
| Department |
Microbiology and Immunobiology
|
| Lab |
CBDM
|
| Street address |
77 Avenue Louis Pasteur
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE194231 |
Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells [CUT&TAG] |
| GSE194253 |
Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells |
|
| Relations |
| BioSample |
SAMN25208996 |
| SRA |
SRX13876433 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5831257_MEC_Pou2f3_R7.CPM.bigwig |
282.7 Kb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
