title: GSM6118829: bone marrow, males from uninfected mothers, rep1, mRNA-derived cDNA; Mus musculus; RNA-Seq
library_construction_protocol: Femurs were harvested from pups either born to S. mansoni infected mothers or uninfected mothers at 28-35 days old. The end of the bone connected to the knee is cut, exposing bone marrow. Using a needle, a hole was punched into the bottom of a 200ul tube, and the bone is placed inside, cut side down, with 50ul of media. This tube was then placed in a 1.5mL tube and then spun at <10,000 RPM for 15 seconds at 4 degrees. Excess media was discarded, and cells were lysed with BD lysis buffer diluted to 1X for 30 seconds. The reaction was quenched using 1%FBS and cells were pelleted by centrifugation at 200xg for 5 minutes at 4 degrees. Primary antibody staining was done for 30 minutes in the dark at 4 degrees and washed twice before being filtered through a 40um filter and pooled. At least 3 mice were used per pool. Then the cells were sorted on a FACS Aria. After sorting, cells were stained with TotalSeq antibodies per manufacture's instruction. Cells were counted and resuspended at a concentration of 10,000 cells/ul before proceeding to library construction. Library was prepared using Chromium Single Cell 5ʹ v2 protocol (10x Genomics). Cellular mRNA and antibody-derived oligos were reverse-transcribed and amplified by PCR according to the Chromium Single Cell 5' v2 protocol (10x Genomics) and with specific primers amplifying the antibody-derived tags. For all cDNA library preparations, we further proceeded according to the manufacturer's instructions for Single Cell 5' v2 protocol (10x Genomics). mRNA and ADT sequences were combined in Cell Ranger, outputting one file per sample for those two libraries. V(D)J library: Library was prepared using Chromium Single Cell 5ʹ v2 protocol (10x Genomics) CITEseq library: Library was prepared using Chromium Single Cell 5ʹ v2 protocol (10x Genomics)