HuBMAP | Digestion and scRNA Analysis of Skin
Arivarasan Karunamurthy, HJ Park
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Abstract
This method details digestion and scRNA analysis of the HuBMAP tissue skin specimens.
Steps
The fresh skin sample is digested enzymatically using Miltenyi Biotec Whole Skin Dissociation Kit, human for 2 hours and further dispersed using the Miltenyi gentleMACS Octo Dissociator.
The resulting cell suspensions are filtered through 70 µm cell strainers twice and resuspended in phosphate-buffered saline (PBS) containing 0.04% BSA.
Resulting cell suspensions are then loaded into 10× Genomics Chromium instrument (Pleasanton, CA) for library preparation. V1 and V2 single-cell chemistries were used per manufacturer’s protocol.
The resultant libraries are sequenced (~200 million reads/sample), using the Illumina NextSeq-500 platform.
The output sequencing reads were then examined by quality metrics, transcripts mapped to reference human genome (GRCh38) and assigned to individual cells according to cell barcodes, using Cell Ranger (10× Genomics).
