title: BaMIPKO_scRNA_islet
design: Age-matched adult mice (8-12 weeks of age) were subjected to tamoxifen injection. Tamoxifen (Cayman Chemical, 10540-29-1, USA) was dissolved in corn oil (Sigma, C8267) at 20 mg/ml and administrated to mice through IP injection at 100 mg/kg body weight for five consecutive days.Islets were isolated 4 months post-tamoxifen injection. In brief, 150-200 mouse islets were isolated and dissociated into single cells (~2.5  105 cells) by TrypLE (Thermo Fisher, 12604013). Approximately 1  105/sample of resuspended single cells were loaded onto microwell-containing agarose plates. Barcoded beads were loaded after cell doublets removal. Cells were lysed using cold lysis buffer (0.1 M Tris-HCl pH 7.5, 0.5 M LiCl, 1% SDS, 10 mM EDTA, and 5 mM dithiothreitol). The beads were then collected and washed once with 1 mL of 6  SSC, once with 500 L of 6  SSC, and once with 200 L of 50 mM Tris-HCl pH 8.0. Samples were then conducted reverse transcription following the Smart-seq2 protocol. The obtained cDNAs were treated with exonuclease I (NEB) and amplified using HiFi HotStart Readymix (Kapa Biosystems). Amplified cDNA libraries were then purified with AMPure XP beads (Beckman Coulter), fragmented, and PCR amplified following the instruction of TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme), replacing the index 2 primers. PCR products were purified with AMPure XP beads (Beckman Coulter) and were subjected to sequencing on the Illumina HiSeq systems by Annoroad Gene Technology (Beijing, China).
identifiers.namespace: SUB15513558
identifiers.id: BaMIPKO_scRNA_islet