GSM9755613: Muscle multiplexed scRNA-seq (naive, onedose, predose2, twodoses), CSP; Mus musculus; RNA-Seq
dLNs were collected, cut into small pieces and digested in RPMI medium containing 100 µg/ml of Dnase I, 1% FCS, and 0.13 U/ml of liberase TM for 30 minutes under agitation at 37ºC. Digestion was stopped by adding 10 mM EDTA and icubating on ice. Cells were filtered using a 70 µm nylon cell strainer, washed, and resuspended in FACS buffe prior to FACS sorting.  Muscle tissue was collected, cut into small pieces, and digested in  RMPI medium containing 1% FCS, 100 µg/ml of DNase I, and 0.1 U/ml of liberase TM for 1 hour at 37ºC under agitation. Preparations were filtered using a 100 µm nylon cell strainer and hematopoietic cells were enriched by centrifugation on a percoll. Cells were washed, cell-surface stained 30 minutes at 4ºC, and resuspended in FACS buffer prior to cell FACS sorting. Library construction was performed according to the manufacturer's instructions (Chromium GEM-X Single Cell v4). Briefly, cells 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 (GEMs). Within each GEM, poly-A RNA and cell-surface hashtag oligonucleotides were reverse transcribed, incorporating a unique 10x barcode and a Unique Molecular Identifier (UMI). The pooled sDNA and feature barcodes constructs were cleaned up with Silane DynaBeads and amplified by PCR. After amplification, the full-length cDNA and smaller hashtag oligo fragments were size-selected to generate independent Gene Expression (GEX) and Cell Surface Protein (CSP) library fractions. During subsequent library construction for both fractions, Illumina R1 and R2 primer sequences, paired-end constructs with P5 and P7 sequences, and unique dual sample indexes were added prior to sequencing.