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Sample GSM7085339

Query DataSets for GSM7085339

Status

Public on Mar 21, 2023

Title

Young_MSC_scRNAseq_16

Sample type

SRA

Source name

Bone marrow

Organism

Mus musculus

Characteristics

cell type: CD45-Ter119-CD31-LepR+CD140a+ mesenchymal stem cell
tissue: Bone marrow
strain: C57BL/6J
age: 9 week old

Extracted molecule

total RNA

Extraction protocol

Young and old MSCs (CD45-Ter119-CD31-CD140a+LepR+) were isolated with a FACSAria II cell sorter from 10-week-old and 2-year-old mice, respectively. Single-cell cDNA synthesis was performed with a C1 Single-Cell Auto Prep System (Fluidigm)
Reverse transcription and preamplification were performed according to the manufacturer’s instructions using a SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (Clontech) and a C1 Single-Cell Auto Prep Reagent Kit for mRNA Seq (Fluidigm). For RNA controls, tubes #1, #4, and #7 of ArrayControl RNA Spikes (Thermo Fisher Scientific) were mixed at a ratio of 100:10:1, and the RNA Spikes mixture was diluted 1:10000 in C1 Loading Reagent (Fluidigm). Sorted MSCs were diluted in Suspension Reagent to ~1000 cells/µL and loaded into a C1-Single-Cell Auto Prep IFC for mRNA-seq (5-10 μm). After loading the cells, the integrated fluidic circuit (IFC) was observed microscopically using an IN Cell Analyzer 6000 (GE Healthcare) to ensure that single cells were captured in each well. Wells with 0, >1, or shrunken cells were excluded. A total of 83 of the 96 wells for young MSCs and 79 of the 96 wells for old MSCs were considered valid. The thermal cycling conditions for cell lysis, reverse transcription, and preamplification were as follows: 72°C for 3 min, 4°C for 10 min, and 25°C for 10 min for cell lysis; 42°C for 90 min and 70°C for 10 min for reverse transcription; 95°C for 1 min and 5 cycles at 95°C for 20 s, 58°C for 4 min, and 68°C for 6 min, 9 cycles at 95°C for 20 s, 64°C for 30 s, and 68°C for 6 min, 7 cycles at 95°C for 30 s, 64°C for 30 s, and 68°C for 7 min, and 1 cycle at 72°C for 10 min for preamplification; and holding at 4°C until harvest. Amplified cDNA (approximately 3.5 μL) from each well was diluted with 10 μL of C1 DNA Dilution Reagent (Fluidigm) and frozen at -30°C until use. The sample in each well was further diluted 1:2 with C1 Harvest Reagent and subjected to tagmentation and Illumina sequencing library preparation using a Nextera XT DNA Sample Preparation Kit (Illumina) and a Nextera XT DNA Sample Preparation Index Kit (96 indices, Illumina). Then, 2.5 μL of Tagment DNA Buffer, 1.25 μL of Amplification Tagment Mix and 1.25 μL of the diluted cDNA sample were mixed and incubated at 55°C for 10 min in a thermal cycler. The tagmentation reaction was then stopped by adding 1.25 μL of neutralizing buffer (NT buffer). Next, the samples were subjected to PCR amplification. A 6.25 μL aliquot of each sample was mixed with NPM buffer, and 1.25 μL each of Index Primer 1 and Index Primer 2. The thermal cycling conditions were as follows: 72°C for 3 min, 95°C for 30 s, 12 cycles at 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 1 cycle at 72°C for 5 min. A 1 μL aliquot from each well of amplified DNA was processed by two rounds of mixing and cleanup with AMPure XP beads (Beckman Coulter), and DNA was then eluted with 144 μL of DNA suspension buffer.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Single cell RNA-seq of bone marrow CD45-Ter119-CD31-LepR+CD140a+ mesenchymal stem cell from young mice

Data processing

Sequencing was performed using an Illumina HiSeq2000 system. Sequence reads were aligned to the murine genome mm9 using RNA-seq Unified Mapper (RUM) version 2.0.4, and transcripts were assembled using Cufflinks version 2.1.1.
Assembly: mm9
Supplementary files format and content: Tab-separated values files and matrix files

Submission date

Mar 07, 2023

Last update date

Mar 21, 2023

Contact name

Keiyo Takubo

Organization name

National Center for Global Health and Medicine

Department

Department of Stem Cell Biology

Street address

1-21-1 Toyama Shinjuku-ku