GSM8957641: CD45+ choroid plexus cells from Tgfbr2-flox: Lyz2-Cre mice; Mus musculus; RNA-Seq
Each sequenced sample contains a pool of 10 mice, 5 males and 5 females, 8-12 weeks old. Upon transcardial perfusion, the brain was harvested and placed into a 10 cm Petri dish with ice-cold PBS, and the choroid plexus (ChP) from the fourth and lateral ventricles were dissected using surgical magnifier glasses. Briefly, the hindbrain was resected using a scalpel, and the fourth ventricle was exposed by gently separating the cerebellum and midbrain with forceps. The ChP from the fourth ventricle was then carefully removed using curved dissection forceps. Next, the brain was bisected sagittally along the midline with a scalpel. The lateral ventricles were exposed by separating the cortex from the midbrain and unfolding the hippocampus. The ChP from the lateral ventricles was dissected from both hemispheres using curved dissection forceps. The ChP was then transferred into a 5 ml Eppendorf tube with ice-cold Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 0.5% Bovine Serum Albumin (BSA, Rockland) and 2.5 mM EDTA (Corning). The dissected ChP was mechanically homogenized with a 3 ml syringe, using 20- and 23-gauge needles in sequence. The choroid plexus cell suspension was filtered through a 30 μm strainer, collected into 5 ml FACS tubes, and spun down at 450g for 5 min. Cell pellets were stained with Fc block and CD45-PE (1:200, Biolegend) for 30 minutes. For live/dead discrimination, a 1 mg/ml of DAPI (Sigma) at 1:5000 final dilution was added to the sample immediately prior to sorting. The DAPI negative, CD45+ population was sorted on FACS Aria-III (BD Bioscience) using a 85µm nozzle. Cells were collected into a 1.5ml Eppendorf tube containing PBS + 0.4% UltraPure BSA (Thermo Fisher) + 1U/µl of Recombinant RNasin Ribonuclease Inhibitor (Promega). Sorted cells were washed in PBS and resuspended into PBS + 0.04% UltraPure BSA buffer at a final volume of 35µl. Single-cell libraries were prepared using the Chromium GEM-X Single Cell 3' v4 protocol, targeting 20,000 cells/sample. Briefly, cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads, and cDNA concentration was determined on the Bioanalyzer. GEX libraries were prepared as recommended by the Chromium GEM-X - Single Cell 3' Reagent Kits v4 (CG000731) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium GEM-X Single Cell 3' Kit v4 - 16 rxns (PN-1000691), Chromium GEM-X Single Cell 3' Chip Kit v4 - 4 chips (PN-1000690) and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq X Plus instrument. Normalized libraries were sequenced on a NovaSeq X Plus Flow Cell using the 151x10x10x151 sequencing recipe according to manufacturer protocol. Read 1 was trimmed to the 10x Genomics recommendation of 28bp. A median sequencing depth of 50,000 reads/cell was targeted for each gene expression library.