HEK293T_Mock_39C_Rep1
Purified total RNA samples were first examined on Bioanalyzer 2100 using RNA 6000 Pico chip to evaluate the integrity and concentration (Agilent Technologies, CA), then normalized onto 96-well plates.  The poly-A containing RNA transcripts in these samples were converted to cDNA using barcoded oligo-dT primers in the reverse transcription reaction to index each sample following the drop-seq method (Macosko EZ et al, Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. 2015; 161(5):1202-1214). The pooled barcoded cDNA samples were amplified by PCR and purified, then turned into sequencing libraries using the Illumina Tagment DNA Enzyme and Buffer kit (Illumina, CA) to include only the poly-A tail adjacent 3 ends of RNA transcripts.  These libraries were examined on the Bioanalyzer (Agilent, CA) DNA HS chips for size distribution and quantified by Qubit fluorometer (Invitrogen, CA), then sequenced on Illumina NovaSeq 6000 S Prime flowcell using the 100 cycle v1.5 kit.  Raw sequencing reads were filtered by Illumina NovaSeq Control Software and only the Pass-Filter (PF) reads were used for further analysis.