GSM6257093: I4; Homo sapiens; RNA-Seq
Approximately 8 mm3 of tissue was cut from each tumor. Samples were incubated at room temperature in 1 ml homogenization buffer for 3 minutes, then dissociated with 13 strokes of a tight pestle in a 2 ml glass homogenizer. Samples were then filtered with a 40 µm mesh filter followed by a 30 µm mesh filter. After brief tabletop centrifugation, the nuclei pellet was resuspended in 1 ml PBA-BSA 0.01% and counted. Nuclei were defined by shape, size, and general appearance, and diluted to 100 nuclei/µl. Single-nucleus RNA-seq library preparation was performed as previously described in Hu, P. et al., Mol Cel, 2017  using a Drop-seq microfluidic system. The quality of the cDNA libraries was evaluated with a bioanalyzer and quantified using a KAPA Library Preparation Kit (Roche Sequencing, cat. # KK4824). Libraries were sequenced using an Illumina NextSeq 500 on high output mode with 20 bp (read 1) and 60 bp (read 2) paired end reads.