title: GSM8714462: single nucleus RNA sequencing S08; Homo sapiens; RNA-Seq
library_construction_protocol: Residual material from the sural nerve grafts were used for control samples in this study. In PNP patients, sural nerve biopsy was performed as part of the clinical diagnostic workup and a small portion of the nerve (approximately 0.2 cm) was used for this study. In Münster and Essen, the samples were immediately fresh-frozen after surgery in dry ice-cooled methylbutane and stored in liquid nitrogen (0 - 14 months) until nuclei extraction. In Würzburg, the samples were embedded into OCT embedding matrix (Roth) until nuclei extraction (33 - 77 months). In Essen and Münster samples were collected from newly recruited patients. In Würzburg, samples were collected from an existing biobank of OCT embedded cryo-preserved sural nerve biopsies collected between 2016 and 2020. For each patient, approximately 10 mg pieces (median of 13.9 mg) were cut on dry ice as starting material from the original samples. Nerve pieces were cut into smaller pieces and processed using the Miltenyi Biotec nuclei extraction protocol. In summary, samples were transferred to a gentleMACS C-tube (Cat.no. 130-093-237) containing 2 ml lysis buffer (nuclei extraction buffer (Cat. no. 130-128-024) + 0.2 U/µl RNase inhibitor (EO0381) and processed on a gentleMACS dissociator with program 4C_nuclei_1. After dissociation, the nuclei suspension was filtered through a pluriStrainer Mini 70-µm cell strainer (pluriSelect®) and washed in 200-400 µl resuspension buffer (PBS with 0.1% BSA and 0.2 U/μL RNase inhibitor (EO0381) depending on sample size and expected nuclei count. Nuclei suspension was then filtered through a pluriStrainer Mini 40-μm cell strainer (pluriSelect®), placed on a 1.5 ml DNA-LoBind tube (Merck). The nuclei suspension was stained with Trypan Blue to manually assess nuclei viability and count using a Fuchs-Rosenthal chamber. Equal volumes between samples were used for downstream application of single nucleus RNA seq. Single nucleus suspensions were loaded into a Chromium Next GEM Chip G and placed into the Chromium X Single Cell Controller and processed with Chromium Next GEM Single Cell 3´ Kit v3.1 reagents (all 10X Genomics). Sequencing was performed on Illumina Nextseq 2000 and Novaseq 6000 with a 28-8-0-91 read setup. CellBender v0.3.0 was used to remove background noise. In CellBender, the number of expected cells and total droplets was based on the UMI curve and the learning rate was reduced based on the automated output report.