GSM5194911: rat DRG_A1; Rattus norvegicus; RNA-Seq
Whole DRG's were dissociated from rat with enzyme mix (Papain, Collagenase) to a single cell suspension, washed in 1X PBS+0.2%BSA solution, passed through a 40-micron cell strainer and FACS sorted using live/dead staining. For human DRG sample L4 lumbar DRG was removed from a 44 year old Asian female. The tissue was immediately snap-frozen and stored at -80C. For nuclei isolation, the entire DRG was processed into smaller pieces by cryopulverization using the CryoPrep. Tissue was then homogenized using EZ Nuclei Lysis Buffer with 0.5% RNasin, 0.5% SUPERase-In and 1mM DTT  or with CST buffer (NaCl2 146 mM, Tris HCl pH 7,5 10 mM, CaCl2 1mM, MgCl2 21 mM, 0.49% CHAP, 0.01% BSA, 0.5% SUPERasin-in, 0.5% RNasin Plus). The homogenate was filtered through a 50-micron filter. The samples were  centrifuged at 500g, 4ºC for 5 mins. The supernatant was removed and the pellet was resuspended in 1 mL of Nuclei Resuspension Buffer (NRB) consisting of 1x PBS, 1% Bovine Serum Albumin and 1% SUPERas-In followed by centrifugation. The final pellet was resuspended in 0.5 mL of NRB containing 6 μM DAPI and filtered through a 20 μm filter. Fluorescence Activated Nuclear Sorting (FANS) was performed to purify nuclei from debris on a FACSAria II (BD). single-cell RNA-Seq libraries were prepared using GemCode Single-Cell 3′ Gel Bead and Library Kit (10x Genomics). polyA selection based RNA capture as per the manufacturer's instructions provided by 10X genomics platform.  Libraries were prepared as per manufacturer's insturctions provided by 10X genomics platform (https://www.10xgenomics.com/single-cell/). libraries were then constructed using standard instrutions of the 10x plaform.