GSM8478767: Huh7, Asibi-[17D-Ens/2A] - rep1; Homo sapiens; RNA-Seq
Sorted cells were then centrifuged and subjected to RNA extraction using RecoverAll Total Nucleic Acid Isolation Kit (Thermo Scientific) according to manufacturer instructions with slight modifications. Briefly, cell pellets were resuspended in digestion buffer and incubated at 50 °C for 3 hrs with constant shaking. During column purification, in-column DNase treatment was omitted. Instead, eluted RNA were treated with TURBO™ DNase (Thermo Scientific) for 30 mins at 37 °C and cleaned up with 0.8X RNAClean XP Beads (Beckman Coulter) . The RNA integrity was assessed by Agilent TapeStation 4200 (Agilent) with High Sensitivity RNA ScreenTape (Agilent). Total RNA samples (300ng per sample) were used as input for reverse transcription using barcoded oligo-dT primers to label each sample in the 96-well plate following the drop-seq method. The pooled barcoded cDNA samples were amplified by PCR and purified, then turned into sequencing libraries using the Tn5 transpose-based tagmentation method to include only the poly-A tail adjacent 3' ends of transcripts.  These libraries were sequenced on Illumina NovaSeq 6000 SP 100 cycle flowcell. Raw sequencing reads were filtered by Illumina NovaSeq Control Software and only the Pass-Filter (PF) reads were used for further analysis.