GSM7063444: Zbtb46 KD MCEC cells, rep2; Mus musculus; RNA-Seq
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation.  Library preparation was performed with 5 to 10ug of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.