title: GSM6170876: bone marrow, males born to control females, rep1, ADT and mRNA-derived combined cDNA; Mus musculus; OTHER
library_construction_protocol: Femurs were harvested from pups 28-35 days old or females chronically infected with Schistosoma mansoni or age-matched control females. 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 AbSeq antibodies per manufacture's instruction. Cells were counted and resuspended at a concentration of 20,000 cells/ul before proceeding to library construction. Library was prepared using BD Rhapsody library construction protocol (BD Biosciences). Cellular mRNA and antibody-derived oligos were reverse-transcribed and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR according to the BD Rhapsody library construction protocol (BD Biosciences) and with specific primers amplifying the antibody-derived tags. SPRI bead size selection was performed in order to separate both the mRNA-derived cDNA (>300bp) and the antibody-derived tagged cDNAs (180bp). For the mRNA derived cDNA library preparation, we further proceeded according to the manufacturer's instructions for BD Rhapsody library construction protocol (BD Biosciences). For antibody-derived tagged library, we used the protocol according to  BD Rhapsody library construction protocol (BD Biosciences) -CITE-seq library: amplification was performed at 95°C for 5min followed by eight cycles at 95°C for 30s; 60°C for 30's; 72°C for 30's and final elongation 72°C for 1min.  Libraries were run on a BioAnalyzer to ensure proper amplification.