GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1684382

Query DataSets for GSM1684382

Status

Public on Aug 19, 2015

Title

Lgr5-EGFP positive cells Replicate 1

Sample type

SRA

Source name

Randomly extracted ex vivo isolated Lgr5 positive cells

Organism

Mus musculus

Characteristics

strain/background: C57BL/6
genotype/variation: Lgr5-EGFP
tissue: small intestinal crypts
cell type: Lgr5-positive

Growth protocol

Crypts were isolated from mouse small intestine, pelleted and mixed with Matrigel (BD). After polymerisation of the Matrigel, growth medium (Advanced DMEM/F12, supplemented with Glutamine, Pennicillin/Streptomycin, Hepes, B27-supplement, N-acetylcysteine, human EGF, RspoI and Noggin) was added. Organoids were passed weekly, with dilution of 1:4.

Extracted molecule

total RNA

Extraction protocol

Organoids were dissociated into single-cell suspension using TrypleE enzyme mix. The cells were pelleted and resuspended in F12 medium with 5 to 10% serum. Primary cells were harvested from freshly isolated small intestines. Crypts were released using PBS0/EDTA followed by Trypsin dissociation. Single cells were FACS-sorted into 96-well plates, containing 100ul Trizol reagent and 0.03ul of 1:50.000 diluted Spike-In RNA. Total RNA was isolated according to the manufacturer's instructions, with the following alterations. RNA was precipitated overnight at -20 °C, with 2ug glycogen.
Single cells were processed using the previously described CEL-seq technique, with a few alterations. A 4bp random barcode as unique molecular identifier (UMI) was added to the primer in between the cell specific barcode and the poly T stretch. RNA pellets were dissolved in primer mix and incubated for 2 minutes at 70 °C. Libraries were sequenced on an Illumina HighSeq 2500 using 50bp paired end sequencing or Illumina NextSeq 500 using 75bp paired end sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

Sample Name: Lgr5SC_1

Data processing

Paired end reads were aligned to the transcriptome using bwaPaired end reads obtained by CEL-seq were aligned to the transcriptome using bwa (version 0.6.2-r126) with default parameters. The transcriptome contained all RefSeq gene models based on the mouse genome release mm10 downloaded from the UCSC genome browser and contained 31,109 isoforms derived from 23,480 gene loci. All isoforms of the same gene were merged to a single gene locus. The right mate of each read pair was mapped to the ensemble of all gene loci and to the set of 92 ERCC spike-ins in sense direction. Reads mapping to multiple loci were discarded. The left read contains the barcode information: the first eight bases correspond to the cell specific barcode followed by 4 bases representing the unique molecular identifier. The remainder of the left read contains a polyT stretch followed by few (<15 transcript derived bases). The left read was not used for quantification. For each cell barcode we counted the number of unique molecular identifiers for every transcript and aggregated this number across all transcripts derived from the same gene locus. Based on binomial statistics we converted the number of observed unique molecular identifiers into transcript counts.
genome build: mm10
processed data files format and content: GSE62270_data_counts_Lgr5SC.txt: Tab separated data file listing all genes (rows) and the number of sequenced transcripts for all samples. Column name indicate the number of the experiment (the 96-well plate cells were sorted into) and the cell barcode number, separated by an underscore. The first column lists the official gene symbol followed by the chromosome name, separated by a double underscore.

Submission date

May 13, 2015

Last update date

May 15, 2019

Contact name

Dominic Grün

E-mail(s)

gruen@ie-freiburg.mpg.de

Phone

+491791073352

Organization name

Max Planck Institute of Immunobiology and Epigenetics