|Public on Feb 16, 2017
|Single cell from pancreas, YFPpos_8w_cell39
time (weeks after arx and dnmt1 depletion): 8
cell type: endocrine pancreatic islet cell
inferred cell type: alpha-like
|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.
|Illumina NextSeq 500
|Single cell from mouse pancreas
|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.
Supplementary_files_format_and_content: tab-delimited text files with raw read values for each sample
|Mar 23, 2016
|Last update date
|May 15, 2019
|James H. Clark Center, 318 Campus Drive,
|Converting adult pancreatic α-cells into β-cells by targeting Dnmt1 and Arx