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Sample GSM3264720 Query DataSets for GSM3264720
Status Public on Jul 01, 2021
Title EKHT1_ROW37_1
Sample type SRA
 
Source name primary somatosensory cortex
Organism Mus musculus
Characteristics chip_col: 1
chip_row: 37
num_input_read: 698437
num_mapped_read: 399836
pct_mt: 12.0%
neuron_type: M1
strain: C57BL6/J
Extracted molecule polyA RNA
Extraction protocol The primary somatosensory cortex was microdissected as aforementioned. Cells were further dissociated by incubating micro-dissected samples in 0.5 mg/mL pronase (Sigma, #P5147) at 37°C for 10 minutes, followed by incubation in 5% bovine serum albumin for 3 minutes and manual trituration in ACSF using pulled glass pipettes. Cells were then centrifuged for 10 minutes at 600 rpm and resuspended before filtration using a 70 mm cell strainer (ClearLine, # 141379C). Cells were then incubated for 10 minutes at 37°C with Hoechst (0.1 mg/mL) and isolated using a Beckman Coulter Moflo Astrios FAC-sorter. Singlet Hoechst+cells were sorted according to their Forward and Side scattering properties (FSC and SSC), and their negativity for Draq7TM (Viability dye, Far red DNA intercalating agent, Beckman Coulter, #B25595). 5000 to 10 000 cells were FACsorted for each experiment. Three microliters of C1 Suspension Reagent (Fluidigm) was added to 10 ml of FAC-sorted cells GFP+ cells, yielding a 300-500 cells / ml suspension. 12 ml of this suspension mix were loaded on a C1 Single-Cell AutoPrep integrated fluidic circuit (IFC) designed for 10 to 17 mm diameter-cells (Fluidigm, #100-5780). RNA was extracted using an RNeasy kit (Qiagen, #74034), and quality control was done using 2001 Bioanalyzer from Agilent. cDNA libraries were obtained using SMARTseq v4 kit (Clontech, # 634888) and sequenced using HiSeq 2500 sequencer.
SMARTseq v4 kit (Clontech, # 634888)
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing read1 containing umi sequences are appended at the end of the headers of read2
read2 are mapped with tophat v2.0.13 on the mouse genome GRCm38 considering Ensembl78 genes annotations
bam files are processed with umi_tools to deduplicate reads with identical UMI sequence
gene expression are quantifyied using summarizeOverlaps() method of the R package GenomicsAlignments(). Only exonic reads are considered in the quantification and this includes 5' and 3' UTRs.
Genome_build: GRCm38
Supplementary_files_format_and_content: retrobeads.tsv.gz contains the result of gene expression quantification by summarizeOverlaps() for each cell. The integers values represent the number of exonic reads mapping into each gene after UMI correction. The first columns contains information about the genes that are quantified.
 
Submission date Jul 11, 2018
Last update date Jul 01, 2021
Contact name Julien Prados
E-mail(s) julien.prados@unige.ch
Phone +41 22 37 95 396
Organization name University of Geneva
Department SCMU
Lab Bioinformatics Support Platform
Street address Rue Michel Servet 1
City Geneva
ZIP/Postal code 1211
Country Switzerland
 
Platform ID GPL17021
Series (1)
GSE116944 Temporal controls over interareal cortical projection neuron fate diversity (retrobeads)
Relations
BioSample SAMN09642808
SRA SRX4380152

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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