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| Status |
Public on Feb 25, 2024 |
| Title |
INA6 multiple myeloma cells without co-culture of HS5 stromal cells [RNA-Seq] |
| Sample type |
SRA |
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| Source name |
Hematopoietic
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Hematopoietic
cell line: INA6
cell type: Plasma Cell
genotype: 46XY, NRAS p.Gly12Asp, TP53 p.Lys132Met , TP53 p.Gln331Ter
treatment: None
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| 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.
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| 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.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were counted and viability was assessed. CD138+ plasma cells were then purified using anti-CD138 microbeads (Miltenyi Biotech, Auburn, United States). CD138+ multiple myeloma cells were then aliquoted separately to perform RNA extraction. RNA was extracted from CD138+ multiple myeloma cells using the RNeasy MiniKit (Qiagen, Germantown, United States). RNA quantity was evaluated using the Qubit RNA Assay Kit (Life Technologies, Carlsbad, United States) and RNA quality was determined on the Bioanalyzer using the RNA Pico Kit (Agilent, Santa Clara, United States). We used at least 500 ng of total RNA for each sample.
Next, library preparation was done with NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, United States), was converted into a DNA library following the manufacturer’s protocol. Library quantity was determined using the Qubit High Sensitivity DNA Kit and library size was determined using the Bioanalyzer High Sensitivity Chip Kit (Agilent). Finally, libraries were put through quantitative PCR using the Universal Library Quantification Kit for Illumina (Kapa Biosystems, Wilmington, United States) and run on the 7900HT Fast quantitative PCR machine (ABI, Grand Island, NY). Libraries passing quality control were diluted to 2 nM using sterile water, and then sequenced
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
RNAseq_INA6
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| 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 (STAR v2.5.4b)
Perform post-alignment quality control (FastQC v0.11.9)
Call transcripts (featureCounts v2.0.1)
Assembly: hg38
Supplementary files format and content: Tab-delimited text files include raw transcript counts for each sample
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| Submission date |
Dec 05, 2022 |
| Last update date |
Feb 25, 2024 |
| Contact name |
Mehmet K. Samur |
| E-mail(s) |
mehmet_samur@dfci.harvard.edu
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Department of Medical Oncology
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| Lab |
Munshi Laboratory
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| Street address |
440 Brookline Avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE220143 |
Bone Marrow Stromal Cell Induced Chromatin Remodeling and Associated Transcriptional Changes in Multiple Myeloma [RNA-Seq] |
| GSE220144 |
Bone Marrow Stromal Cell Induced Chromatin Remodeling and Associated Transcriptional Changes in Multiple Myeloma |
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| Relations |
| BioSample |
SAMN32061226 |
| SRA |
SRX18501013 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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