GSM8527740: Skin, PN2 replicate 4; Rattus norvegicus; RNA-Seq
Single-cell suspensions were prepared from PN2 hippocampus, hypothalamus, bone marrow, and skin and PN7 hippocampus and hypothalamus as described above using a 37% Percoll gradient to purify immune cells. Three to five independent biological replicates were prepared from each condition, with each replicate containing pooled cells from 6 separate animals. We sorted mast cells using a BD Aria II cytometer (courtesy of UMB Flow Cytometry Core), gated on SSC/FSC, singlets, live cells, CD45hi, CD11b-, and FcERI+ in each tissue 1,000-10,000 cells per sample were collected into 350 μL RLT lysis buffer (Qiagen) containing 2-mercaptoethanol per manufacturer's instructions and stored at -80 °C until further processing. RNA was extracted using the Qiagen RNeasy Micro kit. RNA-seq library preparation and sequencing were performed by the Maryland Genomics core facility within the Institute for Genome Sciences. Ultra-low input strand specific RNA-Seq libraries were produced with the SMART-Seq v4 UltraLow Input RNA Kit for Sequencing (Takara, Japan). cDNA was made according to the manufacturer protocol with no modifications. Adapters for sequencing were added using the Illumina Nextera XT library preparation kit (Illumina, San Diego, CA). Input amount was 9 nanograms. Libraries were sequenced using an Illumina NovaSeq 6000 run, targeting 60-70 million read pairs per sample.