GSM8330256: BM_MNX1_IgG_4; Mus musculus; OTHER
Bone marrow cells from MNX1-induced leukemic mice, non-transplanted mouse fetal liver cells containing MNX1-vector as well as FLCs with empty vector were lysed using a in a resuspension buffer containing 0.1% of NP40 and Tween-20 and 0.01% digitonin. Lysed cells were resuspended in transposition buffer (10 mM Tris-acetate, pH 7.6, 5 mM MgCl2, 10% dimethylformamide, 30% PBS, 0.01% digitonin and 0.1% Tween-20) together with Tn5 transposome and tagementation were allowed to proceed for 30 min at 37°C under agitation. For ACT-seq, targeting antibodies used for the pA-Tn5 transposome-antibody complexes were directed against H3K4me3 (Abcam_ab8580), H3K4me1 (Abcam_ab8895), HA-tag (Sigma_H6908),  IgG (Merck Millipore PP64B) and yeast histone H2B (M3930, Boster Bio. Tech., Pleasanton, CA, USA), the latter complexes being used for spike-in to enable sequence read normalization between biological replicates. Sequencing libraries were generated by addition of 25 µl NEBNext High Fidelity 2× Master Mix (NEB), each 2.5 µl of 10 µM primer Tn5mCP1n and Tn5mCBar 7 and 0.5 µl of 100x SYBRGreen I using a LightCycler 480 real-time machine with 72 °C for 5 min, 98 °C for 30 sec and cycling at 98 °C, 10 sec; 63 °C, 30 sec; 72 °C, 30 sec. Amplification was stopped when an increase of about 10 fluorescence units was observed, usually between cycle 12 and 15. . Libraries were bead-purified with 70 µl HighPrep beads and eluted with 12 µl EB (Qiagen). Concentrations and fragment lengths were determined with the Qubit dsDNA High sensitivity kit (Invitrogen) and the TapeStation D1000 High sensitivity assay (Agilent), respectively. Up to ten libraries with compatible barcodes were pooled equimolarly, and pools of 5-10 nM were sequenced in the DKFZ Genomics and Proteomics Core Facility (GPCF) using an Illumina NextSeq 550 system with 75 bp paired-end, mid output mode.