title: GSM2670764: NOD_islet_3wk_2; Mus musculus; RNA-Seq
library_construction_protocol: Islets were harvested as described previously (Calderon et al., 2015) and dispersed using Cell Dissociation Solution Non-Enzymatic (Sigma-Aldrich, St. Louis, MO) for 5 min at 37°C. Pancreatic lymph nodes and lungs were harvested, finely chopped with scissors, placed in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA) containing 125 ug/mL Liberase TL (Roche Life Sciences, Indianapolis, IN) and 50 ug/mL DNase I, grade II (Roche) and incubated at 37°C for 30 minutes and dispersed via gentle agitation. Single-cell suspensions incubated in PBS, pH 7.4 (Life Technologies) having 1% bovine serum albumin (BSA, Sigma), and 50% of 2.4G2 conditioned DMEM at 4°C for 15 min to block Fc receptors. Cells were stained with fluorescently labeled antibodies and sorted on a FACSAria II or analyzed on the FACSCanto II (BD Biosciences). Macrophages were sorted based on their expression of CD45, F4/80 and I-Ag7. DC were selected based on their expression of XCR1. In all cases CD3e+ and CD19+ events were eliminated from the sort (dump gated). For cell sorting experiments, approximately 500-5000 macrophages were isolated from 6-10 mice and this represented 1 biological sample in our analysis. At least 3 samples were used for each population studied by RNAseq analysis. All flow cytometry data was analyzed using FlowJo software version 10.2 (Tree Star Software, Ashland, OR). Sorted cell RNA was isolated using the Ambion RNAqueous-Micro Kit (Life Technologies) following the manufacturer’s instructions Library preparation was performed with 1-50 ng of total RNA, integrity was determined using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA).  ds-cDNA was prepared using the SeqPlex RNA Amplification Kit (Sigma) per manufacturer’s protocol.  cDNA was blunt ended, had an adenosine added to the 3’ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-2500 using single reads extending 50 bases. All RNAseq work was performed by the Genome Technology Access Center at Washington University School of Medicine.
attributes.tag: GEO Accession
attributes.value: GSM2670764