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Sample GSM7670878

Query DataSets for GSM7670878

Status

Public on Aug 05, 2023

Title

CT9w, Control-1

Sample type

SRA

Source name

kidney

Organism

Mus musculus

Characteristics

tissue: kidney
treatment: Untreated

Extracted molecule

nuclear RNA

Extraction protocol

kidney samples of cortex with outer medulla were cut into <8mm3 pieces and homogenized in 2 ml of ice-cold Nuclei EZ Lysis buffer (NUC101; Sigma) with protease inhibitor (5892791001; Roche) and RNase inhibitor (N2615, Promega; AM2696, Life Technologies) using a Dounce homogenizer (885302-0002; Kimble Chase). The homogenate was further incubated on ice for 5 min with an additional 2 ml of lysis buffer. Then the homogenate was filtered through a 40-μm cell strainer (43-50040-51; pluriSelect) followed by centrifugation at 500 × g for 5 min at 4°C. The pellet was resuspended and washed with 4 ml of the buffer and incubated on ice for another 5 min. After another centrifugation, the pellet was resuspended in Nuclei Suspension Buffer (1x PBS, 1% bovine serum albumin, 0.1% RNase inhibitor) and filtered through a 5-μm cell strainer (43-50005; pluriSelect).
Nuclei were processed using Chromium Single Cell Controller and Chromium Next GEM Single Cell 3’ Reagent kit v3.1 (10x Genomics, Pleasanton, CA) according to the manufacturer’s protocol. Briefly, 3,000-4,000 nuclei were loaded onto the Chromium Controller to generate single cell GEMs coated with unique 10x cell barcodes, unique molecular identifiers (UMI), and poly dT oligos. RNA molecules from single nuclei were reverse-transcribed within droplets to generate barcoded cDNAs. After break emulsion, cDNA molecules were pre-amplified, fragmented, ligated to an adaptor, and amplified with sample indices, followed by a double-sided size selection using SPRI beads (Beckman Coulter). The final libraries were assessed by Bioanalyzer 2100 (Agilent) and Qubit HS DNA (ThermoFisher) and pooled.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

PT Features(Cis9w vs CT9w)-1sta.cvs

Data processing

The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software 5.0.1
Assembly: mm10
Supplementary files format and content: Comma Separated Values (CSV)file and matrix files

Submission date

Aug 01, 2023

Last update date

Aug 05, 2023

Contact name

Zhengwei Ma

E-mail(s)

zma@augusta.edu

Organization name

Augusta University

Street address

1460 Laney walker Blvd.