title: GSM8017738: UI; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow cells were collected from the C3H/HeN mice, pooled, and centrifuged at 500xg for 5 minutes at 4°C. Supernatant was discarded. Cell pellet was resuspended in 4 mL of 0.14M Ammonium Chloride solution to lyse RBC. After 5 minutes incubation at RT, RBC lysis was stopped by adding 8 mL of fresh R10. Centrifuged at 500xg for 5 minutes at 4°C. Supernatant was discarded. Cells were resuspended in 40 mL of R10+15%LCCM and passed through 70 µM cell strainer. 10 mL of cell suspension was plated in each petri dish and incubated at 37°C. After 4 days, 70% media was replaced with fresh R10 media and incubated at 37°C. After 8 days of initial plating, the culture media was removed and washed with 10 mL of cold PBS twice and 2 mL of Accutase™ Cell Dissociation Reagent (Stem cell) was added, incubated at 37°C. After 20 minutes, plates were tapped well to detach the cells completely from the plate. BMDM cell suspension was supplemented with 4 mL of fresh R10 media and centrifuged at 500xg for 5 minutes. Supernatant was discarded. The cells were resuspended at 1x106 cells/mL concentration in fresh R10 media without Pen/Strep. In a non-treated 6-well plate, 1x106 BMDM were plated in each well. After overnight incubation, the cells were infected with 1 mL of BbLP (1 µg/mL) resuspended in R10 media. PBS treated BMDM was maintained as control. After 1 or 4 hpi, Cells were removed by accutase, washed with cold PBS and the viability of the cells were checked using Trypan Blue and counted using a hemocytometer. After viability check, the cells were used for RNA isolation. 10x Genomics single cell libraries were generated by the University of Texas at San Antonio (UTSA) Genomics Core. Cell suspensions were loaded into 10x Genomics microfluidics chips and onto the 10x Chromium to capture single cells in Gel Beads-in-emulsion (GEMs) reactions. 5000 cells/sample were targeted for capture in GEMs preceding library prep following manufacture's recommendations for the 3' Gene Expression v3 kit.