NextSeq 500 paired end sequencing; GSM2096686: mouse_YFPpos_8w_cell1; Mus musculus; RNA-Seq

mouse_YFPpos_8w_cell1

After their destruction in adult mice, insulin-producing pancreatic beta-cells slowly regenerate from other islet cells, like glucagon-producing alpha-cells. However the molecular basis of this conversion is unknown. Moreover it remains unclear if this intra-islet cell conversion is relevant to human diseases with extensive beta-cell loss, like in type 1 diabetes (T1D). Here, we show that subsets of glucagon-expressing cells in subjects with T1D produce Insulin and other molecular features of beta-cells, accompanied by loss of the alpha-cell regulators DNA methyltransferase 1 (Dnmt1) and Aristaless-related homeobox (Arx). We generated mice permitting lineage tracing and inactivation of Dnmt1 and Arx in adult alpha-cells. Within 3 months of Dnmt1 and Arx loss, 50% of alpha-cells converted into cells producing insulin protein but not glucagon, changes not observed in alpha-cells after only Arx or Dnmt1 loss. Single cell isolation and high-throughput RNA sequencing revealed efficient and extensive alpha-cell conversion into progeny indistinguishable by global gene expression from native beta-cells. Our work reveals pathways regulated by Arx and Dnmt1 sufficient for achieving targeted generation of beta-cells from adult pancreatic alpha-cells. Overall design: Single-cell RNA-seq of in-vivo conversion of pancreatic a-cells into ß-cells

Converting adult pancreatic a-cells into ß-cells by targeting Dnmt1 and Arx

Submitted by Gene Expression Omnibus on 17-FEB-2017

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.

Converting adult pancreatic a-cells into ß-cells by targeting Dnmt1 and Arx

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