GSM5268093: 4267_WT-6M-Ki67-; Mus musculus; RNA-Seq
Briefly, to lyse the sorted cells, the samples were thawed at RT, 75µl of PKD buffer was added and incubated for 1 h at 56 °C at 300 rpm for reverse cross-linking.  Samples were then centrifuged at 20000g for 20 min.  For RNA extraction, the miRNeasy FFPE Kit was used following manufacturer's instructions to obtain 14 µl of RNA.  Samples were quantified using a High Sensitivity D5000 DNA assay on an Agilent 2200 Tapestation. Purified full-length cDNA was processed to NGS libraries using the Nextera XT DNA library preparation and the Nextera XT Index kits (Illumina). In short, cDNA was diluted to 200 pg/µl, 100 pg were tagmented. Tagmentation was terminated and primer mix and buffers were added and tagmented cDNA was PCR amplified. The finished NGS libraries were purified using Ampure XP Beads at a 1:1 ratio and eluted in 15 µl of nuclease-free water before quantification and QC using a High Sensitivity D5000 DNA assay on an Agilent 2200 Tapestation. Nextseq500 high output chemistry V1, 75 bp SR or 75 bp & 75 bp PE