GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM7899309

Query DataSets for GSM7899309

Status

Public on Mar 05, 2024

Title

PanSci, mouse ileum, part 5

Sample type

SRA

Source name

ileum

Organism

Mus musculus

Characteristics

tissue: ileum
genotype: C57BL/6, B10

Extracted molecule

nuclear RNA

Extraction protocol

The 10X PBS-hypotonic stock solution was prepared as per the method outlined in [doi: 10.1038/s41596-022-00752-0]. On the day of nuclei extraction, fresh working lysis solution was prepared by diluting 1X hypotonic lysis buffer with RNase-free water (Corning, #46-000-CM), supplementing with 3mM MgCl2, 1% Diethyl pyrocarbonate (Sigma Aldrich, #40718) and specifically optimized detergent for each organ/tissue type: 0.025% IGEPAL CA-630 (VWR, #IC0219859650) for kidney, lung, liver, brown adipose tissue, inguinal adipose tissue, and perigonadal adipose tissue; 0.01% Digitonin (Thermo Fisher, #BN2006) for heart, muscle, duodenum, jejunum, ileum, and colon; 0.49% CHAPS (Sigma Aldrich, #220201) for the stomach. Additionally, 0.33M sucrose (Sigma Aldrich, #S0389) was included in the working lysis buffer for the stomach and intestinal tissues (duodenum, jejunum, ileum, and colon) to maintain osmotic balance and protect the nuclei. Dry tissue powder stored in liquid nitrogen was directly transferred into 10 mL of the pre-prepared working lysis solution, and a brief vortexing for 10 seconds is followed to disperse larger powder chunks. The mixture was then incubated at 4 °C for 15 minutes with constant racking, and immediately passed through a 40 μm cell strainers (VWR, #470236-276) with the 5 mL syringe plunger. Filter was then rinsed with an additional 5 mL working lysis solution. Finally the extracted nuclei were collected by centrifugation at 500g for 5 minutes at 4 °C, and resuspended in nuclei suspension buffer [10 mM Tris-HCl pH 7.5 (Thermo Fisher, #15567027), 10 mM NaCl (Thermo Fisher, #AM9760G), 3 mM MgCl2 (Sigma Aldrich, #68475-100ML-F) supplemented with 1% SUPERase⋅In RNase Inhibitor (Thermo Fisher, #AM2696) and 0.2mg/mL BSA or Recombinant Albumin (New England Biolabs, #B9000S or #B9200S)] supplemented with 0.005 mg/mL DAPI (Thermo Fisher, #D1306) for fluorescence-activated cell sorting. Sorting were perform on SH800 Cell Sorter with 100 μm sorting chip (Sony, #LE-C3210). All DAPI positive singlet nuclei were included to unbiased recover global cell population and remove cellular debris and doublet cells. Nuclei were collected into a 1.5mL tube (Eppendorf, #022431021 ) with a 100 μL nuclei suspension buffer as the receiving buffer. Sorted clean nuclei were then collected by centrifugation 500g for 5 minutes at 4 °C.
EasySci-RNA; The sequencing library generation for the sorted nuclei was conducted in accordance with the EasySci protocol [doi: https://doi.org/10.1101/2022.09.28.509825]. Briefly, the sorted nuclei were first distributed into 96-well plates for reverse transcription, where indexed oligo-dT primers and indexed random hexamers were used to add the first index. After this, the nuclei were then pooled, washed, and redistributed into fresh 96-well plates for the attachment of a second index by ligation. Following another pooling and washing step, the nuclei were redistributed into the final plates for second-strand synthesis, purification, tagmentation with Tn5 transposase and finally PCR to add the final indexes. Final PCR product is pooled and purified with 0.8X AMPure XP SPRI Reagent (Beckman coulter, #A63882). Each library's quality was assessed using an Agilent TapeStation and sequencing was performed on an Illumina NovaSeq 6000 System with an S4 Flow Cell. For the current study, in addition to the previously reported mouse brain data, a total of 25 NovaSeq S4 runs were executed to generate the new data.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

EXP115_Ileum
PanSci_ileum_genecount.mtx
PanSci_ileum_df_cell.csv
PanSci_ileum_df_gene.csv

Data processing

Demultiplex - bcl2fastq/v2.19.0.316 tolerating one mismatched base in barcodes (edit distance (ED) < 2)
Trim - trim_galore/v0.4.1 with default settings
Alignment - STAR/v2.5.2b with default settings
SAM spilt and gene count generation - customized scripts (https://github.com/JunyueCaoLab/EasySci)
Assembly: mm39
Supplementary files format and content: genecount.mtx - gene count sparse matrix file for each organ
Supplementary files format and content: df_cell.csv - cell annotation csv file for each organ
Supplementary files format and content: df_gene.csv - gene annotation csv file for each organ

Submission date

Nov 14, 2023

Last update date

Mar 05, 2024

Contact name

Zehao Zhang

E-mail(s)

zzhang03@rockefeller.edu

Organization name

The Rockefeller University

Lab

Laboratory of single-cell genomics and population dynamics