Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: 4 weeks following tamoxifen, spinal cords were dissected and enzymatically dissociated with 2mg/ml collagenase-4 for 1 hour at 37 oC. Cell suspensions were subsequenty strained and centrifuged before removing debris (Mytlenyi debris removal kit). Liberated single cells were re-suspended in 1% bovine serum album in HBSS before FACS purification. For FACS purification, forward and side scatter gates were used to exclude debris, and cell doublets, then tdTomato cells were purified. Libraries were prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v2 Chemistry as per the manufacturer's protocol. In brief, single cells were sorted into 1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed first using Illumina MiSeq SR50 to approximate the number of recovered cells in each sample.