title: GSM6250303: SD, replicate 2, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Schwann cells (SCs) were isolated from the sciatic nerves of 23-week-old SD and HFD mice following a modification of the previously described protocol by Wolbert et al.  (PMID: 32295886). Briefly, sciatic nerves were extracted from mice intracardially perfused with Hank's buffered salt solution (HBSS, Invitrogen ThermoFisher, Waltham, MA). Nerve segments were finely chopped, incubated in HBSS at 37oC, and then digested in Dulbecco's Modified Eagles Medium (DMEM, Lonza, Basel, Switzerland) containing trypsin (Gibco ThermoFisher, Waltham, MA; 0.25%) and collagenase II (Worthington Biochemical, Freehold, NJ; 1.62 U/μl) at 37°C for 20 minutes. Cells were triturated using fire-polished Pasteur pipettes, filtered through a 70-μm cell strainer (Falcon, Ontario, Canada), and centrifuged at 300 rpm for 10 minutes. They were then re-suspended in 120 μl of phosphate buffered saline (PBS) supplemented with 5% fetal bovine serum (FBS) and 20 μl of Anti-O4 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) to ensure selective purification of SCs. SC magnetic enrichment step was performed as per the manufacturer's instructions. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, SCs 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-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina 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 SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.