The female B10.RIII mouse was purchased from The Jackson Laboratory (ME, USA, strain #: 000457). The animal was maintained under specific-pathogen free (SPF) conditions and euthanized at 13 weeks of age by Carbon dioxide (CO2) inhalation. Splenocytes were isolated using 1X PBS (Gibco), ACK lysing Buffer (Quality Biological), FCS serum (HyClone), RPMI medium (HyClone), DNA Stabilizer (Bionano Genomics), and Cell Buffer (Bionano Genomics). Single cell suspension of 6.8 million Splenocytes were used for high molecular weight genomic DNA (HMW gDNA) extraction, using MagAttract HMW DNA Kit from Qiagen (Cat. No. 67563) for PacBio. Ultra-high molecular weight (UHMW) gDNA was extracted from mouse splenocytes with the Bionano Prep SP Blood and Cell DNA Isolation Kit (Bionano Genomics, 80030) as described in the Bionano Prep SP Frozen Cell Pellet DNA Isolation Protocol (Bionano Genomics). Brifely, 5 million frozen pelleted cells were thawed in a 37oC bath and resuspended in DNA Stabilizing Buffer (Bionano Genomics). After that, the cells were lysed in the presence of detergents, proteinase K, and RNase A and the UHMW gDNA was bound to a silica Nanobind Disk (Bionano Genomics), washed, and eluted. The extracted gDNA was equilibrated for 3 days at room temperature to homogenize and quantified with Qubit dsDNA BR assay kit (Thermo Fisher Scientific, Q32850) and Femto Pulse (Agilent).
strain
B10.RIII
age
13 weeks
sex
female
tissue
Spleen
biomaterial_provider
Rachel Caspi, National Institutes of Health, National Eye Institute, Laboratory of Immunology, Immunoregulation Section, 9000 Rockville Pike, Bldg. 10, Room 10S243, Bethesda, Maryland 20892, USA
birth_date
7/27/21
birth_location
The Jackson Laboratory
cell_type
Splenocytes
collected_by
Yingyos Jittayasothorn
collection_date
2021-10-26
health_state
Healthy
sample_type
Freshly isolated splenocytes
treatment
No
BioSampleModel
Model organism or animal