Mar 21, 2024
HuBMAP | Digestion and scRNA Analysis of Skin
- Arivarasan Karunamurthy1,
- HJ Park1
- 1University of Pittsburgh Medical Center
Protocol Citation: Arivarasan Karunamurthy, HJ Park 2024. HuBMAP | Digestion and scRNA Analysis of Skin. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62krzgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 21, 2024
Last Modified: March 22, 2024
Protocol Integer ID: 97072
Keywords: digestion, scRNA
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Abstract
This method details digestion and scRNA analysis of the HuBMAP tissue skin specimens.
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).