|
| Status |
Public on Feb 25, 2024 |
| Title |
MM1S multiple myeloma cells co-cultured with HS5 stromal cells for 72 hours |
| Sample type |
SRA |
| |
|
| Source name |
Hematopoietic
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Hematopoietic
cell line: MM1S
cell type: Plasma Cell
genotype: 45XX, KRAS p.Gly12Ala, TRAF3 p.Val536_Asn545delValPheValAlaGlnThrValLeuGluAsninsAsp
treatment: Co-Culture with HS5 bone marrow stromal cells
|
| Treatment protocol |
For co-culture experiments, the multiple myeloma cell lines and HS5 were passaged and cultured separately for 24 hours in a 75 cm2 flask. After 24 hours of culture and once HS5 50% confluency was observed, the multiple myeloma cells were manually counted and 10 million multiple myeloma cells with 90% or greater viability (assessed using Tryptan Blue staining), were transposed into 20 ml of fresh RPMI-1640 medium and either added into a 75 cm2 flask containing HS5 cells or cultured alone in a new flask. After 72 hours of culture, total cells were collected from each condition using adherent culture protocol and Trypsin-EDTA to remove attached cells.
|
| Growth protocol |
The human multiple myeloma cell lines MM.1S, RPMI-8226, INA-6, and the human stromal cell line HS5 were cultured in RPMI-1640 medium supplemented with complete medium (10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine) at 37°C and 5% CO2. Recombinant human IL-6 at a concentration of 1 ng/mL (R&D Systems, Minneapolis, United States) was added to INA-6 culture medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each condition, 50,000 CD138+ multiple myeloma cells were aliquoted and prepared in duplicates as previously reported.46 Cells were lysed for 10 minutes at 4°C in lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-360). After lysis, the pellets were subject to a transposition reaction (at 37°C for 60 minutes) using the TD buffer and transposase enzyme (Illumina Nextera DNA preparation kit, FC-121–1030). The transposition mixture was purified using a MinElute PCR purification kit (Qiagen).
Library amplification was performed using custom Nextera primers and the number of total cycles determined by running a SYBR-dye based qPCR reaction. Amplified libraries were purified using a PCR purification kit (Qiagen) and sequenced.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Perform pre-alignment quality control (FastQC v0.11.9)
Trim reads for quality (Trim Galore v0.5.0)
Adapter removal (Cutadapt v1.18)
Align to GRCh38 reference genome (bowtie2 v22.4.1)
Perform post-alignment quality control (FastQC v0.11.9)
Assembly: Call peaks (MACS2 v2.2.7.1)
Supplementary files format and content: hg38
Supplementary files format and content: bigWig, narrowPeak
|
| |
|
| Submission date |
Dec 05, 2022 |
| Last update date |
Feb 26, 2024 |
| Contact name |
Mehmet K. Samur |
| E-mail(s) |
mehmet_samur@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Department of Medical Oncology
|
| Lab |
Munshi Laboratory
|
| Street address |
440 Brookline Avenue
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE220142 |
Bone Marrow Stromal Cell Induced Chromatin Remodeling and Associated Transcriptional Changes in Multiple Myeloma [ATAC-Seq] |
| GSE220144 |
Bone Marrow Stromal Cell Induced Chromatin Remodeling and Associated Transcriptional Changes in Multiple Myeloma |
|
| Relations |
| BioSample |
SAMN32060879 |
| SRA |
SRX18500562 |
