GSM8443272: AZFa_KO_2; Mus musculus; RNA-Seq
On ice, around 10mg of frozen testis was homogenised in 1ml of lysis buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 8, 1% BSA, 0.1% IGEPAL and freshly added 1 µM DTT, 0.4 U µl−1 RNase Inhibitor (New England BioLabs), 0.2 U µl−1 SUPERasIn (ThermoFischer Scientific)) and incubated on ice for 4 minutes. The lysate was filtered through a 30µm filter and then centrifuged at 500g for 5 minutes at 4C. The supernatant was removed, and the pellet resuspended in 1ml of wash buffer 1 (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 8, 1% BSA, and freshly added 1 µM DTT, 0.4 U µl−1 RNase Inhibitor, 0.2 U µl−1 SUPERasIn). This was spun down at 500g for 5 minutes at 4°C and washing step repeated. Next the pellet was resuspended in 670 µl of freshly diluted dithio-bis (succinimidyl propionate) (DSP) (1mg/ml in PBS) and incubated at room temperature for 30 minutes. Fixation was quenched by adding 14.07µl Tris-HCL, pH8. Nuclei were spined down at 500g for 5 minutes at 4C and resuspended in 1ml of wash buffer 2 (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10mM Tris-HCL, pH8, 1% BSA, and freshly added 1 µM DTT, 0.4 U µl−1 RNase Inhibitor, 0.2 U µl−1 SUPERasIn). The sample were spun down one more time at 500g for 4 minutes at 4°C and the pellet resuspended in 0.04% BSA in PBS.  Testis nuclei isolation was adapted from a previously published protocol36. On ice, around 10mg of frozen testis was homogenised in 1ml of lysis buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 8, 1% BSA, 0.1% IGEPAL and freshly added 1 µM DTT, 0.4 U µl−1 RNase Inhibitor (New England BioLabs), 0.2 U µl−1 SUPERasIn (ThermoFischer Scientific)) and incubated on ice for 4 minutes. The lysate was filtered through a 30µm filter and then centrifuged at 500g for 5 minutes at 4C. The supernatant was removed, and the pellet resuspended in 1ml of wash buffer 1 (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 8, 1% BSA, and freshly added 1 µM DTT, 0.4 U µl−1 RNase Inhibitor, 0.2 U µl−1 SUPERasIn). This was spun down at 500g for 5 minutes at 4°C and washing step repeated. Next the pellet was resuspended in 670 µl of freshly diluted dithio-bis (succinimidyl propionate) (DSP) (1mg/ml in PBS) and incubated at room temperature for 30 minutes. Fixation was quenched by adding 14.07µl Tris-HCL, pH8. Nuclei were spined down at 500g for 5 minutes at 4C and resuspended in 1ml of wash buffer 2 (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10mM Tris-HCL, pH8, 1% BSA, and freshly added 1 µM DTT, 0.4 U µl−1 RNase Inhibitor, 0.2 U µl−1 SUPERasIn). The sample were spun down one more time at 500g for 4 minutes at 4°C and the pellet resuspended in 0.04% BSA in PBS. Libraries were prepared according to the manufacturers protocol from 10X Genomics for 10X_3prime_mRNA