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Sample GSM2096714 Query DataSets for GSM2096714
Status Public on Feb 16, 2017
Title mouse_YFPpos_8w_cell29
Sample type SRA
Source name Single cell from pancreas, YFPpos_8w_cell29
Organism Mus musculus
Characteristics facs_yfp_signal: YFPpos
time (weeks after arx and dnmt1 depletion): 8
cell type: endocrine pancreatic islet cell
inferred cell type: delta-like
Extracted molecule total RNA
Extraction protocol Isolated mouse islets were dissociated into single cells by enzymatic digestion using 0.05% Trypsin (25300-120; Life Technologies). 7-aminoactinomycin D (7-AAD, eBiosciences) was used at 1:500 dilution as Live/Dead stain to exclude dead cells. Sort gates were adjusted with reference to negative controls (wild-type islets without YFP labeling). We collected cells from YFP+, lineage-traced population and also YFPNeg, non-labeled population. Cells were sorted on a special order 5-laser FACS Aria II (BD Biosciences) using a 100 µm nozzle at a flow rate of 1 following doublet removal. Sorted single-cells were collected directly into 96-well plates (Bio-Rad cat #: HSP9601) containing 4 µL of lysis buffer with dNTPs37 for downstream single-cell RNA-Seq assays.
Single-cells were collected in lysis buffer in 96-well plates, followed by reverse transcription with template-switch using an LNA-modified template switch oligo to generate cDNA. After 21 cycles of pre-amplification, DNA was purified and analyzed on an automated Fragment Analyzer (Advanced Analytical). Each cell’s cDNA fragment profile was individually inspected and only wells with successful amplification products (concentration higher than 0.06 ng/ul) and with no detectable RNA degradation were selected for final library preparation. Tagmentation assays and barcoded sequencing libraries were prepared using Nextera XT kit (FC-131-1024; Illumina) according to the manufacturer’s instructions. Barcoded libraries were pooled and subjected to 75 bp paired-end sequencing on the Illumina NextSeq instrument.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description Single cell from mouse pancreas
Data processing Sequencing reads were trimmed, adapter sequences removed and the reads aligned using STAR with default parameters.
Duplicate reads were removed using picard. Transcript counts were obtained using HT-Seq and mm10 UCSC exon/transcript annotations.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files with raw read values for each sample
Submission date Mar 23, 2016
Last update date May 15, 2019
Contact name Martin Enge
Organization name Stanford University
Department Bioengineering
Lab Stephen Quake
Street address James H. Clark Center, 318 Campus Drive,
City Stanford
State/province CA
ZIP/Postal code 94305
Country USA
Platform ID GPL19057
Series (1)
GSE79457 Converting adult pancreatic α-cells into β-cells by targeting Dnmt1 and Arx
BioSample SAMN04575255
SRA SRX1656005

Supplementary file Size Download File type/resource
GSM2096714_B2-1000200103.csv.gz 71.8 Kb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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