title: GSM5252954: PreL_IgG_Cut&Tag; Mus musculus; OTHER
library_construction_protocol: Murine spermatocytes were isolated by FACS as described previously (Gaysinskaya, Soh, van der Heijden, & Bortvin, 2014; Getun, Torres, & Bois, 2011) with modification. Wild-type or mutant mice of different ages were used for isolation of spermatocytes at different stages: 7-11 days old mice were used for isolating Sertoli, spermatogonia, and preleptotene cells, 2-week old mice for zygotene cells, 3- to 5-week-old mice for pachytene, diplotene, and Metaphase II cells, and 1-2 months old SUN1 W151R mutant mice for zygotene-like spermatocytes CUT&Tag libraries for Sertoli, pre-leptotene, leptotene/zygotene and pachytene/diplotene cells were generated as previously described(Kaya-Okur et al., 2019) with Hyperactive In-Situ ChIP Library Prep Kit for Illumina kit (Vazyme Biotech, TD901). Briefly, 50k cells for each CUT&Tag library were incubated with 10μl balanced concanavalin A–coated magnetic beads at room temperature for 10 min. The bead-bound cells were resuspended with 50μl of antibody buffer. 1 microgram anti-CTCF(Rabbit monoclonal to CTCF,  Abcam, ab128873) antibody  and Rabbit IgG(Yeasen, 36113ES10) was then added to the bead-bound cells. After 2-hour incubation at room temperature, the primary antibody was discarded carefully and 0.5μl secondary antibody diluted with 50μl Dig-wash Buffer was added to the cells. The cells were then incubated at room temperature for 1 hour. Afterwards, samples were washed gently with 800μl Dig-wash Buffer, and 0.58μl pG–Tn5 together with 100μl Dig-300 buffer was added to the samples. After 1-hour incubate at room temperature, samples were washed gently with 800μl Dig-300 Buffer. 300μl Tagmentation Buffer was then added to each sample and the samples were incubated at 37 °C for 1 h. The interactions were quenched by adding 10μl 0.5M EDTA,3μl 10% SDS and 2.5μl 20 mg/ml Proteinase K and incubating the samples at 50 °C for 1 hour. Phenol-chloroform and ethanol precipitation, as well as PCR and size-selection were performed as described in Hi-C library construction. All CUT&Tag libraries were sequenced on Illumina Nova Seq 6000 platform at PE150 mode subsequently.
attributes.tag: GEO Accession
attributes.value: GSM5252954