Library preparation and sequencing steps follow the same steps as in the DropSeq method (version 3.1 dated 28 December 2015, http://mccarrolllab.com/dropseq/) (17 (link)) and SeqWell method (23 (link)) where composition of buffers, sequence of primers used, and protocol steps are described in extended detail. Briefly, the beads were first washed with 6× SSC twice after retrieval, and the captured mRNA was reverse-transcribed using Maxima H Minus reverse transcriptase (ThermoFisher) with a custom template switching oligo (AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG). The cDNA coated beads were then treated with Exonuclease I (Exo I, NEB) for 45 min at 37°C to chew away any unbound mRNA capture probes. The beads coated with cDNA was then amplified using a half PCR reaction (i.e. only 13–16 cycles rather than 35–40), using 13 cycles for cell lines or large cells and 16 cycles for primary cells as in SeqWell method (23 (link)). The amplified DNA was purified using Ampure XP beads (Beckman Coulter) at 0.6 ratio, and the quality of the amplified DNA was assessed by Agilent BioAnalyzer using high sensitivity chip. Purified cDNA was then pooled and inputted for standard Nextera tagmentation and amplification reactions (Nextera XT, Illumina) using a custom primer instead of i5 index primer to amplify only those fragments that contain the cell barcodes and UMIs. The PCR product was then purified using Ampure XP beads at 0.6× ratio, and the quality of the libraries were checked by Agilent BioAnalyzer high sensitivity chip. The libraries were sequenced on HiSeq 2500 sequencer (Illumina) using a custom primer for Read 1 with 75 cycles on Read 1 and 75 cycles on Read2. For Read1, only the first 20 bases were used in analysis. PhiX libraries were used at 20% as spike-in controls.