|
| Status |
Public on Jun 13, 2024 |
| Title |
Vehicle Control A375 Smad2/3 6.11.18 |
| Sample type |
SRA |
| |
|
| Source name |
A375 Cells
|
| Organism |
Homo sapiens |
| Characteristics |
chip antibody: Smad2/3
cell line: A375 Cells
treatment: Vehicle
|
| Treatment protocol |
Cells were starved for 2 hours. Treated with 10ng/mL TGFB1 ligand or 4mM HCl containing 1mg/mL BSA vehicle control for 2 hours.
|
| Growth protocol |
A375s were grown in filter sterilized DMEM, 10% FBS, 1% PenStrep, 1% Glutamine at 37°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A375 cells were fixed directly in 15cm plates with 11% formaldehyde and collected by scraping. Approximately 100,000,000 cells were used per condition. Cells were lysed using lysis buffers with protease inhibitors (Roche #05056489001) and sonicated such that fragmented chromatin was 200-300bp long. Optimal chromatin length was confirmed by gel electrophoresis. Prior to antibody addition, 50µL chromatin was collected for input sample. The remaining sonicated chromatin was incubated overnight at 4°C with 10µg antibody attached to Dynabeads (Invitrogen #10004D). Samples (including inputs) were washed with wash buffers and eluted for 6 hours at 70°C, treated with RNaseA (Sigma #R4642) and ProteinaseK (Life Technologies #AM2546), and purified using Zymo ChIP DNA Concentrator kit (Genesee Scientific #11-379C).
Libraries were end repaired (VWR #ER81050), polyA tailed (Invitrogen #18252-015, NEB #M0212L), adaptor ligated (NEB #E7335), size selected using Ampure XP beads (Beckman Coulter #A63881, Life Technologies #12027), and PCR amplified (NEB #M0531).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
ChIP-Seq aligned to GRCh38 with Bowtie with max mismatch 2bp
peaks were called using MACS 2.0 with the following setting: q-value 0.05
Assembly: GRCh38
Supplementary files format and content: narrowPeak Bed files from MACS 2.0 output
|
| |
|
| Submission date |
Sep 14, 2022 |
| Last update date |
Jun 13, 2024 |
| Contact name |
Leonard Zon |
| E-mail(s) |
zon@enders.tch.harvard.edu
|
| Organization name |
Boston Children's Hospital
|
| Department |
Oncology/Hematology
|
| Street address |
1 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE213326 |
A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma [ChIP-seq] |
| GSE213360 |
A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma |
|
| Relations |
| BioSample |
SAMN30852606 |
| SRA |
SRX17567503 |
