|
| Status |
Public on Nov 13, 2023 |
| Title |
invitro_mtDNA_0uM_TFAM_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
in vitro mtDNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: in vitro mtDNA
mtase conditions: 200 U Hia5 for 10 min at 37C
|
| Treatment protocol |
HSMM differentiation was induced by switching cells at 80% confluency to DMEM containing glucose and pyruvate supplemented with 2% Heat Inactivated Horse Serume (Thermo Fisher Scientific) and 0.4 µg/mL dexamethasone
Mitochondria were isolated by cellular fractionation, permeabilized in Permeabilization Buffer (20 mM Tris, pH 7.4; 70 mM Potassium Acetate; 250 mM Sucrose; 0.25% Tween-20; 0.25% NP-40/Igepal-630). Mitochondria were treated with MTase as above in Reaction Buffer (20 mM Tris, pH 7.4; 70 mM Potassium Acetate; 250 mM Sucrose) and DNA was extracted and subjected to PacBio library construction
|
| Growth protocol |
HeLa S3 cells were maintained in DMEM + 10% Fetal Bovine Serum (FBS) in humidified atmosphere with 5% CO2 at 37C
HSMM cells were maintained in Human Skeletal Muscle Cell Basal Media with growth supplement (Cell Applications, INC)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation. Extracted DNA was treated with RNase A and XmaI. DNA from HeLa cells was linearized with BamHI, and DNA from HSMM with EagI. This was followed by a Phenol:Chloroform:Isoamyl Alcohol extraction, a chloroform:isoamyl alcohol extraction, and a second ethanol precipitation.
PacBio SMRTbell libraries were prepared from linearized DNA (102-182-700)
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Sequel II |
| |
|
| Data processing |
Generate CCS reads (ccs) with HiFi kinetics
Predict m6A positions using HiFi kinetics (fibertools predict-m6a)
Align reads using pbmm2
Extract fiberseq data and generate bed file (fibertools ft extract)
Footprints identified using an HMM (custom scripts available at hhtps://github.com/churchmanlab)
Assembly: Hg38 (aligned to custom modifications of chrM, available at https://github.com/churchmanlab)
Supplementary files format and content: bed formatted mapped molecule locations with ZMW ID in column 4 and methylation locations in column 12
Supplementary files format and content: tsv formatted per-read footprint information with read ID in column 1. Each additional column represents a position in the genome. A positive value indicates a footprint/protection and a negative value indicates accessible DNA. The number indicates the size of the region covering that genomic position
Supplementary files format and content: pq formatted read info file to go along with tsv file. Contains information on individual footprinted reads including ZMW ID, read ID corresponding to TSV, and methylation count.
|
| |
|
| Submission date |
Sep 02, 2022 |
| Last update date |
Nov 13, 2023 |
| Contact name |
Richard Stefan Isaac |
| E-mail(s) |
richard_isaac@hms.harvard.edu
|
| Phone |
8657199109
|
| Organization name |
Harvard Medical School
|
| Department |
Genetics
|
| Lab |
Churchman Lab
|
| Street address |
77 Avenue Louis Pasteur, NRB 356
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL28352 |
| Series (2) |
| GSE212608 |
Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA |
| GSE212611 |
Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA |
|
| Relations |
| BioSample |
SAMN30648660 |
| SRA |
SRX17409009 |
