GSM3615551: KDM2AB_CXXCfl_TAM_nucRNA_r2; Mus musculus; RNA-Seq
1x10^7 mESCs were mixed with 2.5x10^6 Drosophila SG4 cells in PBS. Cells were lysed in 1 ml HS lysis buffer (0.05% NP40, 50mM KCl, 10 mM MgSO4.7H20, 5mM HEPES, 1mM PMSF, 3mM DTT, 1xPIC). Nuclei were recovered by centrifugation at 1000 g for 5min and washed three times in 1 ml resuspension buffer (10mM NaCl, 10mM Tris pH 7.4, 3mM MgCl2). Nuclear integrity was assessed using 0.4% trypan blue staining (ThermoFisher). Nuclear RNA was prepared from 4x10^6 nuclei using TRIzol reagent according to the manufacturer's protocol (Invitrogen), then treated with the TURBO DNA-free kit (ThermoFisher). RNA quality was assessed using the 2100 Bioanalyzer RNA 6000 Pico kit (Agilent), then RNA was depleted of rRNA using the NEBNext rRNA depletion kit. To quantitate the exact SG4 cell: mESC mixing ratio for each individual sample, genomic DNA was isolated from a small aliquot of nuclei by phenol-chloroform extraction. Genomic DNA was then tagmented using Tn5 transposase, and genomic DNA libraries were prepared alongside ATAC-seq libraries, for which ATAC was performed using another aliquot of the same nuclei. Genomic DNA libraries therefore serve as input controls for both cnRNA-seq and cATAC-seq experiments. Further details can be found in the ATACseq SubSeries. RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA-seq kit (NEB), following the manufacturer's protocol and using NEBNext Multiplex Oligos (NEB). Libraries were sequenced using the Illumina NextSeq 500 platform with 80 bp paired-end reads in biological triplicate or quadruplicate.