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Sample GSM6387759

Query DataSets for GSM6387759

Status

Public on May 24, 2023

Title

RBG25050_WT

Sample type

SRA

Source name

Bone marrow

Organism

Mus musculus

Characteristics

cell type: Lin-Sca1+cKit+
tissue: Bone marrow
genotype: WT

Extracted molecule

total RNA

Extraction protocol

To harvest mouse bone marrow cells, spine, femurs, tibiae and pelvic bones were crushed with a pestle and mortar in 3 mL PBS + 2% FBS. Further 3 mL PBS + 2% FCS were used to wash the bones and the resulting cell suspension was filtered through a 0.75 uM filter. Cells were pelleted and resuspended in 3 mL PBS + 2% FBS. Red cell lysis was performed using cold ammonium chloride (StemCell Technologies cat. no 07800). PBS 2% FCS was added to dilute the red cell lysis buffer during subsequent centrifugation at 4 oC. For lineage depletion, EasySep™ Mouse Hematopoietic Progenitor Cell Enrichment Kit from StemCell Technologies (cat no. 19816A) was used according to the manufacturer’s instructions but avoiding the use of rat serum. Samples were transferred into 5 ml polystyrene (352058) FACS tubes and incubated with 10 µl/ml EasySep mouse hematopoietic progenitor cell isolation cocktail labelled with biotin. 40 µl/ml of EasySep™ Streptavidin RapidSpheres were added and allowed to bind on ice for 10 minutes. The samples were subsequently diluted to 2.5 ml using PBS + 2% FBS and placed in the EasyStep magnet (StemCell Technologies cat no. 18000) for 3 minutes at room temperature. After incubation, samples were transferred into new tubes, spun down, and resuspended in 100 ul PBS + 2% FCS. For antibody staining, a master mix was prepared as indicated in table 2 and cell suspensions were incubated on ice, in the dark for 30 minutes. After staining, cells were washed, pelleted, and resuspended in 100 ul PBS + 2% FCS. Were necessary, streptavidin conjugated antibody was added in a second step. Samples were pelleted and resuspended to a final volume of 500 ul PBS + 2% FCS. We used single colour, unstained, and full panel controls that were made using pooled BM cells from all mice before lineage depletion.
Primary single cells (LSK cells – Lin-Sca1+ckit+) were FACS index sorted into lysis buffer (0.2% Triton X-100 (Sigma), RNase inhibitor (SUPERase, Thermofisher), and nuclease-free water (Thermo Fisher)). scRNA sequencing was performed according to the Smart-Seq2 protocol.
Libraries were prepared using the Illumina nextera XT DNA preparation kit and sequenced on the Illumina Hi-Seq 2500.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Smart-Seq2

Data processing

Reads were mapped simultaneously to the Mus musculus genome (mm10) and the 92 ERCC sequences using STAR. FeatureCounts was used to count the number of reads mapped to each gene. Quality control was applied to exclude dead and dying cells as well as poor quality cells (using the parameters fGenes:Total > 12%, mit:mappedToGene < 20%, nNuclearReads > 150K, ercc:mapped < 20%). Data was pre-processed using the in-house pre-processing tool smqpp in Python analysed using Scanpy (1.7.1). Cells were normalised using the DESeq2 method and logged.
Assembly: mm10
Supplementary files format and content: a h5ad file that contains raw counts, metadata for each cell and gene annoataion information.

Submission date

Jul 25, 2022

Last update date

May 24, 2023

Contact name

Xiaonan Wang

Organization name

Shanghai Jiao Tong University School of Medicine

Department

School of Public Health

Street address

227 Chongqing South Road