alias: GSM3376173
title: GSM3376173: DroNcSeq_healthy; Mus musculus; RNA-Seq
library_construction_protocol: [Single cell dissociation protocol] Kidney from a C57BL/6 mouse was minced into 1 mm pieces with a razor blade and incubated at 37 ˚C in enzyme dissociation buffer containing 250 U/ml Liberase (Roche) and 40 U/ml DNase I. After 10 min, the solution was transferred to a Miltenyi C-tube and the gentleMACS D1 program was run (Miltenyi). Cells were further digested for additional 10 min with trituration, and the reaction was stopped by adding 10% fetal bovine serum. The dissociated cells were pelleted by centrifugation (500 g, 5 min), washed twice, passed through a 35 µM FACS tube (Falcon) and pelleted again by centrifugation (500 g, 5 min). Cells were resuspended in PBS+1%BSA and purified by FACS with optimal gates set for side scatter and forward scatter. [Nuclei extraction protocol] Nuclei were isolated with Nuclei EZ Lysis buffer (Sigma #NUC-101) supplemented with protease inhibitor (Roche #5892791001) and RNase inhibitor (Promega #N2615, Life Technologies #AM2696). Samples were cut into <2 mm pieces and homogenized using a Dounce homogenizer (Kimble Chase #885302-0002) in 2ml of ice-cold Nuclei EZ Lysis buffer and incubated on ice for 5 min with an additional 2ml of lysis buffer. The homogenate was filtered through a 40-µm cell strainer (pluriSelect #43-50040-51) and then centrifuged at 500 x for 5 min at 4 ºC. The pellet was resuspended and washed with 4 ml of the buffer and incubated on ice for 5 min. After another centrifugation, the pellet was resuspended with Nuclei Suspension Buffer (1x PBS, 0.07% BSA, 0.1% RNase inhibitor), filtered through a 20-µm cell strainer (pluriSelect 43-50020-50) and counted. We used the inDrops protocol developed by Klein et al to generate single nucleus transcriptome. [Dropseq protocol] We used the DropSeq protocol described by Mackosco et al to generate the single cell transcriptomes [sNuc-Dropseq protocol] We adapted the DropSeq platform to profile single nucleus with the methods reported by Hu et al  (Mol Cell, 2017) [DroNcSeq protocol] We used soft lithography techniques to fabricate the DroNc-seq silicon master microfluidic devices. DroNc was performed as previously described by Habib et al (Nature Methods, 2017) [sNuc-10x protocol] We followed the guideline provided by the manufacturer (10x Genomics) to generate single nucleus transcriptomes RNA libraries were prepared for sequencing using standard Dropseq and 10X Chromium procedure single cell/nucleus RNA-seq
identifiers.namespace: GEO
identifiers.id: GSM3376173