title: GSM9013738: E16.5-CSB-scRNAseq-WT; Mus musculus; RNA-Seq
library_construction_protocol: Prepare and oxygenate ACSF on ice, then make 3% low-melt agarose in ACSF (47°C). Prepare 1× HBSS with 0.04% BSA (Ca²⁺/Mg²⁺-free) and chill, along with Miltenyi tissue digestion solution (37°C). Embed brain tissue in agarose, section at 300 μm, and collect slices in ice-cold ACSF. After washing slices 3× with HBSS to remove divalent cations, isolate CSB tissue, digest in 500 μL enzyme solution (37°C, 15 min with intermittent pipetting), then quench with HBSS and filter. Centrifuge the filtrate (600 rcf, 5 min, 4°C), resuspend cells in 100 μL HBSS, and assess viability via trypan blue before proceeding with 10x Genomics single-cell RNA sequencing. Library was performed according to the manufacter's instructions (single cell 3'v1 protocol, 10xGenomics).Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion(GEM).The poly-ARNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA,which contains an llumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode.The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRI select reagent for subsequent library construction.