title: GSM6094669: Total8; Homo sapiens; RNA-Seq
library_construction_protocol: All living donor samples used for sequencing were processed within one hour of organ retrieval. Biopsies were collected in RPMI 1640 on ice, and mechanically dissociated with a blade before enzymatic digestion at 37ºC with 0.1 mg/ml DNase I, 3300 CDA units/ml Collagenase MA  and 1430 NP units/ml BP neutral protease  for 20 minutes at 37ºC with intermittent agitation. Cell suspensions were filtered through 35µm cell strainer snap-cap FACS tubes and a plunger from a 1ml syringe was used to gently mash remaining tissue in the strainer before rinsing strainer lid with 1:1 volume of FBS on ice. A low frequency (<1%) of immune cells in the single cell suspension from a kidney biopsy core necessitated immune enrichment in 10 samples (5 males, 5 females) using magnetic EasySep Human CD45 depletion kit II as per the manufacturer's modified instructions for positive selection of CD45-expressing cells.  Samples were prepared according to 10X Genomics Single Cell 3' v3 Reagent kit user guide. Samples were washed twice in PBS plus 0.04% BSA, and viability was determined by a hemocytometer via Trypan Blue staining. Following counting, the appropriate volume for each sample was calculated for a target capture of 9,000 cells. For CD45-enriched samples, all cells were sequenced. Samples that were too low in cell concentration as defined by the user guide were washed, re-suspended in a reduced volume and counted again using a haemocytometer prior to loading onto the 10x single cell B chip. After droplet generation, samples were transferred onto a pre-chilled 96 well plate, heat sealed and incubated overnight in a Veriti 96-well thermos cycler. The next day, sample cDNA was recovered using Recovery Agent provided by 10x and subsequently cleaned up using a Silane DynaBead mix as outlined by the user guide. Purified cDNA was amplified for 11 cycles before being cleaned up using SPRIselect beads. Samples were diluted 4:1 (elution buffer (Qiagen):cDNA) and run on a Bioanalyzer (Agilent Technologies) to determine cDNA concentration. cDNA libraries were prepared as outlined by the Single Cell 3' Reagent Kits v3 user guide with modifications to the PCR cycles based on the calculated cDNA concentration. The molarity of each library was calculated based on library size as measured bioanalyzer and qPCR amplification data. Samples were pooled and normalized to 1.5 nM. Library pool was denatured using 0.2N NaOH  for 8 minutes at room temperature, neutralized with 400mM Tris-HCL. Library pool at a final concentration of 300pM were loaded to sequence on Novaseq 6000 (Illumina). Samples were sequenced with the following run parameters: Read 1-28 cycles, Read 2- 90, index 1-10 cycles, index 2-10 cycles. Across samples, cells were sequenced to a target depth of 40,000 reads per cell.