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| Status |
Public on Mar 11, 2024 |
| Title |
WT_BLISS_rep2 |
| Sample type |
SRA |
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| Source name |
Thymus
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| Organism |
Mus musculus |
| Characteristics |
tissue: Thymus
cell type: mTEChi
genotype: WT
age: 4-6 weeks old
Sex: Female
strain: C57BL/6J
treatment: NA
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| Treatment protocol |
mTECs were isolated by collagenase and dispase digestion, CD45+ cells depletion using magnetic beads, fluorescent antibodies staining, and flow sorting.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We followed the suspension-cell BLISS protocol. Cells were fixed in 4% paraformaldehyde (15710, Electron Microscopy Sciences) for 10 min at room temperature on a roller shaker. Formaldehyde was quenched with glycine. Fixed cells were then washed twice using ice-cold PBS. For permeabilization, fixed cells were subjected to two sequential lysis using lysis buffer 1 (10mM Tris-HCl, 10mM NaCl, 1mM EDTA, 0.2% Triton X-100, pH 8) and lysis buffer 2 (10mM Tris-HCl, 150mM NaCl, 1mM EDTA, 0.3% SDS, pH 8). After permeabilization, DSB ends were blunted using the Quick Blunting Kit (E1201L, NEB) for 1 h at room temperature, which was followed by in situ DSB ligation of BLISS adapters using T4 DNA ligase (M0202M, NEB) for ~16 h (overnight) at 16°C. On Day 2, genomic DNAs were reverse crosslinked and extracted using DNA extraction buffer (10mM Tris-HCl, 100mM NaCl, 50mM EDTA, 1% SDS, 1mg/mL Proteinase K, pH 8) at 55°C for ~18 h (overnight) with shaking. On Day 3, DNA was purified and then sonicated in 1 x TE buffer using the Covaris M220 ultrasonicator with the following setting: peak incident power 50, duty factor 10%, cycles per burst 200, time 70s. DNA was then purified by Agencourt AMPure XP beads. DNA was in vitro transcribed using the MEGAscript T7 Transcription Kit (AM1333, ThermoFisher) at 37°C for ~14 h (overnight). On Day 4, genomic DNA was removed using DNase I (RNase-free) (AM2222, ThermoFisher) followed by RNA purification using Agencourt RNAClean XP beads (Beckman Coulter).
Libraries were built via the following steps: Illumina RA3 adapter ligation using T4 RNA Ligase 2, truncated KQ (M0373S, NEB) at 25°C for 2 h; reverse transcription using SuperScript III Reverse Transcriptase (18080044, ThermoFisher) at 50°C for 1 h; library indexing and amplification using NEBNext High-Fidelity 2x PCR Master Mix (M0541S, NEB); DNA purification and size-selection (300-800bp) using Agencourt AMPure XP beads. All primers and adapters were custom synthesized by Integrated DNA Technologies (IDT) based on sequences in TruSeq Small RNA Library Preparation kit (Illumina).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Cutadapt (v2.5) was used to scan BLISS reads for matches of the expected 16bp prefix [8bp unique molecular identifier (UMI) + 8bp sample barcode] in the BLISS adapters, allowing a maximum of one mismatch. Only reads containing the 16bp prefix were kept. Prefixes were clipped using Cutadapt.
Reads were aligned to the corresponding genome using bowtie2 (v2.3.4.3). Only uniquely mapped reads were kept (SAMtools, v1.3.1).
PCR duplicates that had the same UMIs (allowing for a maximum of two mismatch) were removed using UMI-tools (‘dedup’, v1.0.1). DSBs independently generated at the same genomic location in different cells were kept because they had different UMIs.
Bam files were generated using SAMtools (v1.3.1). Bigwig files were generated using deepTools (‘bamCoverage’, v3.0.2).
Assembly: mm10 and CAST/EiJ
Supplementary files format and content: *.bw: bigwig files
Library strategy: Breaks Labeling In Situ and Sequencing (BLISS)
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| Submission date |
Feb 05, 2023 |
| Last update date |
Mar 11, 2024 |
| Contact name |
CBDM Lab |
| E-mail(s) |
cbdm@hms.harvard.edu
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| Phone |
617-432-7747
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| Organization name |
Harvard Medical School
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| Department |
Microbiology and Immunobiology
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| Lab |
CBDM
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| Street address |
77 Avenue Louis Pasteur
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE224552 |
Z-DNA underlies the target choice of Aire by facilitating promoter poising [BLISS] |
| GSE224557 |
Z-DNA underlies the target choice of Aire by facilitating promoter poising |
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| Relations |
| BioSample |
SAMN33097539 |
| SRA |
SRX19285534 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7025876_WT_BLISS_rep2.bw |
39.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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