|
| Status |
Public on Jan 10, 2019 |
| Title |
P03_Ctrl02-38439524_single_cell_passage_03 |
| Sample type |
SRA |
| |
|
| Source name |
single_cell_passage_03
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: healthy donor
cell type: Bone marrow stromal cells (BMSCs)
passage: 3
|
| Treatment protocol |
N/A
|
| Growth protocol |
One vial of frozen Passage 2 BMSCs was thawed and plated in T75 flasks following the SOP. During the serial culture, cells were seeded at 5000 cells per cm2 and harvested at 70-80% confluence. From passage 3 to 8, cells were passaged after 5 days of culture, and culture media was changed on day 3; from passage 9 to 10, the cells were passaged after 8 days of culture, and culture media was changed on day 3 and 6. Cells from each passage were cryopreserved in a solution containing 90% FBS and 10% DMSO for further studies.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen cells from different passages were thawed and plated in T75 flasks for 5-7 days. Once they reached 70-80% confluence, cells were collected for single cell separation. Fluidigm C1 Single-Cell Auto Prep Array for mRNA-Seq (10-17um) and Fluidigm C1 system were used for cell capture following the Fluidigm protocol, then reverse transcription and cDNA amplification were performed using the SMARTer PCR cDNA Synthesis kit (Clontech, version 1) on the C1 unit.
Single-cell cDNA from validation cells were harvested for library preparation. Nextera XT DNA sample preparation kits and Index kits were used for library preparation following the manufacturer’s protocol. Single-cell cDNA libraries from each passage were pooled and cleaned up, then cDNA size distribution was examined by 2100 Bioanalyzer. This was followed by quantification and normalization with the KAPA Library Quantification Kit and BioRad CFX96 qPCR machine (BioRad). Single cell mRNA sequencing was performed on NextSeq 500 by using the NextSeq High Output v2 kit (300 cycles). PhiX Control v3 (Illumina) was used as a quality control for sequencing runs.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
processed data file: single_cell_passage_03_processed.txt
P03-BMSC-Ctrl02_S2
|
| Data processing |
BCL files generated by Illumina NextSeq were converted to standard FASTQ files following Illumina’s protocol (basespace.illumina.com).
STAR - 2.4.1d for alligment
Partek E/M to generate gene counts
Genome_build: hg19-RefSeq Transcripts 2016-5-2
Supplementary_files_format_and_content: gene counts
|
| |
|
| Submission date |
Jan 09, 2019 |
| Last update date |
Jan 10, 2019 |
| Contact name |
Shutong Liu |
| E-mail(s) |
shutongliu@hotmail.com
|
| Organization name |
National Institutes of Health
|
| Department |
Clinical Center
|
| Street address |
9000 Rockville Pike
|
| City |
BETHESDA |
| State/province |
Maryland |
| ZIP/Postal code |
20814 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE124858 |
Single Cell Sequencing Reveals Gene Expression Signatures Associated with Bone Marrow Stromal Cell Subpopulations and Time in Culture [scRNA-seq] |
| GSE124862 |
Single Cell Sequencing Reveals Gene Expression Signatures Associated with Bone Marrow Stromal Cell Subpopulations and Time in Culture |
|
| Relations |
| BioSample |
SAMN10718958 |
| SRA |
SRX5233219 |
