Mus musculus
Protocols: Dissected spleen fragments were transferred to a 40-μm cell strainer (Greiner # 542040) and ground with a syringe plunger. The cell suspension was strained twice. Strainers were washed with additional DMEM (Gibco # 41966029) with 5% FBS and cells were pelleted by centrifugation at 300 x g for 4 minutes at 4°C. Cell pellets were carefully resuspended in 300 uL of ACK lysis buffer (Lonza # BP10-548E) for 1 minute in ice. After incubation cells were washed with 1mL of DMEM with 5% FBS and pelleted. DNA was crosslinked, fragmented, ligated, de-crosslinked and shuffled to get circular templates. Inverse PCR was performed using independent primer sets respectively targeting the 5' end of the chrX duplication (forward TTCAGTAGGTGTGACAGAGA; reverse ACATAATTATAGCGCTGGCA), 3' end of the chrX duplication (forward GCATCTGTCTGCTTACTTTG; reverse GGTCTGGATACCTTTCAAGG) and the chrY translocation site identified with the first two pairs (forward TAAAAGGGGAGGTAGACACA; reverse GTGTTCAAACCAGACTCCT). PCR products were purified, library prepped using the Nextera DNA Flex (Illumina) chemistry and pooled.