GSM8452925: Con, replicate 3, scRNAseq; Mus musculus; RNA-Seq
Peripheral blood samples were collected and gently rotated to mix thoroughly. Whole blood was first diluted by adding an equal amount of sterile phosphate-buffered saline (PBS), the diluted whole blood was layered onto Ficoll PAQUE, after horizontally centrifuged, the middle mononuclear cell layer was transferred to a new tube, ACK lysis buffer (Miltenyi Biotec) was added to lyse red blood cells. After the last wash, pipet off the supernatant and loosen the pellet by adding PBS with 0.04% BSA. Whole transcriptome libraries were prepared using the BD Rhapsody single-cell whole-transcriptome amplification workflow. In brief, second strand cDNA was synthesized, followed by ligation of the WTA adaptor for universal amplification. Eighteen cycles of PCR were used to amplify the adaptor-ligated cDNA products. Sequencing libraries were prepared using random priming PCR of the whole-transcriptome amplification products to enrich the 3′ end of the transcripts linked with the cell label and UMI. The final libraries were quantified using the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific) and the size distribution of the libraries were determined using a High Sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). All libraries were sequenced by illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run.