title: GSM8876174: Bone marrow, MCAO; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow were flushed from tibias and femurs with PBS/1% BSA and filtered through a 40 μm cell strainer and centrifuged at 300g for 5min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Miltenyi Biotec). BD Rhapsody system was used to capture the transcriptomic information of the bone marrow sample-derived single cells. Single-cell capture was achieved by random distribution of a single-cell suspension across > 200,000 microwells through a limited dilution approach. Beads with oligonucleotide barcodes were added to saturation, so that a bead was paired with a cell in a microwell. The cells were lysed in the microwell to hybridize mRNA molecules to capture oligos on the beads. Beads were collected into a single tube for reverse transcription and ExoI digestion. Upon cDNA synthesis, each cDNA molecule was tagged on the 5′ end (that is, the 3′ end of a mRNA transcript) with a unique molecular identifier (UMI) and cell barcode indicating its cell of origin. Whole transcriptome libraries were prepared using the BD Rhapsody single-cell whole-transcriptome amplification (WTA) workflow including random priming and extension (RPE), RPE amplification PCR and WTA index PCR. The libraries were quantified using a High Sensitivity DNA chip (Agilent) on a Bioanalyzer 2200 and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific). Sequencing was performed by Illumina Novaseq6000 (Illumina, San Diego, CA) on a 150 bp paired-end run.
identifiers.namespace: GEO
identifiers.id: GSM8876174_r1