GSM5710225: Lymph node WHIM [LN_WHIM]; Mus musculus; RNA-Seq
Peripheral lymph node stromal cell suspensions were prepared from lymph node slices digested with 900 U/ml Collagenase IV and 40 μg/ml Dnase1 for  30 min at 37°C. Cells from 2 mice per genotype were pooled. CD45 and Ter119+ cell were magnetically depleted. The leftover cells were stained with antibodies against CD45, Ter119, CD71, CD31 and GP38. Lymph node stromal cells were sorted as CD45- Ter119- CD71- CD31- cells into 350 μL DMEM with 20% FBS. Splenic stromal cells suspensions were prepared the same way. Cells from 3 mice per genotype were pooled. CD45 and Ter119+ cell were magnetically depleted.The leftover cells were stained for CD45, Ter119, CD71, LIN (CD3e, B220, CD19), CD31, GP38 and PDGFRa. Splenic stromal cells were sorted as CD45- Ter119- LIN- CD71- CD31- into 350 μL DMEM with 20% FBS. The libraries were prepared using the Chromium Single Cell 3ʹ Reagent Kits v3.0 and run on an Illumina NovaSeq system with 100-bp paired-end reads to a coverage of ~ 65,000 mean reads per cell and > 90% saturation for lymph node stromal cells and to a coverage of ~ 30,000 - 57,000 mean reads per cell and > 80% saturation for splenic stromal cells. The sequencing reads were aligned onto the Mus musculus GRCm38 (mm10) reference genome.