GSM9050451: Uninfected Host Bone Marrow; Mus musculus; RNA-Seq
Bone marrow cells were harvested from naïve and C. difficile-infected mice by flushing cleaned femur and tibia with ice cold phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) and red blood cells were lysed using ACK lysing buffer. Blood was obtained by intra-cardiac puncture from naïve and C. difficile-infected mice and collected in ethylenediaminetetraacetic acid microtainers, followed by an RBC lysis with ACK lysing buffer. For lamina propria leukocyte isolation, approximately 2 cm sections were excised from cecal tissue of C. difficile-infected mice and cleaned by gentle shaking in buffer A (HBSS with 5% FBS and 25mM HEPES) two times. Clean tissue was transferred to Buffer B (HBSS buffer with 2mM EDTA and 25mM HEPES) and vortexed vigorously to remove mucus layer. Samples were then incubated at 37oC shaking water bath in a buffer containing HBSS + 15 mM HEPES + 5 mM EDTA + 10% FBS + 0.015% or 1mM Dithiothreitol. Resulting tissue was mechanically minced and digested enzymatically in Iscove's media containing liberase TL (Roche; 0.17 mg/ml) and DNase (Sigma; 30 ug/ml) for 30 min in a 37oC shaking water bath. Samples underwent further dissociation using gentleMACS™ Dissociator. Samples were resuspended in Buffer B and then filtered with a 100 µm filter. Final cell suspension was prepared in 1 x PBS with 1U/µl of RNase inhibitor. Neutrophils from each tissue were magnetically sorted using MACS MS columns (Miltenyi Biotech #130-097-658, Germany) according to the manufacturer's instructions. Neutrophil sorting buffer was supplemented with 1U/µl of RNase inhibitor. For colon samples, in addition to magnetic beads that were part of the kit, mouse CD326 (EpCAM) MicroBeads were also used to select out EpCAM+ cells and thus reduce the amount of intestinal epithelial cells. Cells were then resuspended in 1 x PBS with 1U/µl RNase inhibitor and 10% FBS at room temperature. Cell viability was determined by trypan blue exclusion. All samples sent for sequencing had viability >70%. Sequencing was performed using Chromium X scRNAseq and Illumina platform equipment at CCHMC Single Cell Genomics Core. For cell preparation, NextGEM assay was used and sequencing with 3'v3.1 assay chemistry. Libraries were prepared using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics) according to the manufacturer's protocol.