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Sample GSM3376173

Query DataSets for GSM3376173

Status

Public on Oct 13, 2018

Title

DroNcSeq_healthy

Sample type

SRA

Source name

Kidney

Organism

Mus musculus

Characteristics

protocol: Single nucleus isolation
disease state (group): Healthy
platform: DroNcSeq

Extracted molecule

total RNA

Extraction 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

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

single nucleus RNA-seq
Processed data file: Healthy.combined.dge.txt.gz

Data processing

We used a newly developed pipeline, zUMIs (Parekh et al., 2018), to process the single cell and single nucleus sequencing data from mouse kidney. In brief, we first filtered out the low-quality barcodes or UMIs based on sequence with the internal read filtering algorithm built in zUMIs. We then used zUMIs to map the filtered reads to mouse reference genome (mm10) using STAR 2.5.3a (two-pass mapping mode). Next, zUMIs quantified the reads uniquely mapped to exonic or intronic reads and inferred the true barcodes that mark cells by fitting a k-dimensional multivariate normal distribution with mclust package. Finally, a UMI count table utilizing both exonic and intronic reads were generated for downstream analysis.
Genome_build: mm10
Supplementary_files_format_and_content: The .txt files report gene-level raw UMI count matrices. The .txt files' column headers contain barcode sequences.

Submission date

Sep 05, 2018

Last update date

Oct 13, 2018

Contact name

Haojia Wu

E-mail(s)

[email protected]

Organization name

Washington University in St. Louis

Department

Renal Division/Internal Medicine