Read1

scRNA-seq libraries were prepared by amplifying the 3′ untranslated region (UTR) of the transcripts by using the modified CEL-Seq2 protocol (Hashimshony et al., 2016 (link)), which replaced the SuperScript II reverse transcriptase with Maxima H minus (EP0752; Thermo Fisher Scientific), the second strand synthesis reagent with the second strand synthesis module (E6111; New England BioLabs). A total of 384 cells in the same plate were pooled after reverse transcription. Each condition was analyzed in triplicate. Sequencing reads were obtained from HiSeq 1500 platform at the following cycles: 15 cycles of Read1 (Unique Molecular Identifier: 6 bp, cell barcode: 9 bp) and 36 cycles of Read2 (Illumina, San Diego, CA, United States), as shown in Supplementary Table S2.

The constructed transpososome was used to tagment about 3 femtograms of yeast genomic DNA (ATCC Cat. No. 9763), which was serially diluted from a stock of 6 ng/μL, to generate a primary library^{13 (link)}. The transposase was removed from DNA by Protease K (New England Biolabs, Cat. No. P8107S) treatment and then Protease K was denatured by heat. The primary library was sequentially amplified by Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific Cat. No. F565L) with NEX8a for three cycles, and then together with Illumina Read1 (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′) and Read2 (5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′) primer pair for 20 cycles, both with 30-min extension time at 65 °C. The amplified library was cleaned with AMPure (Beckman Coulter Cat. No. A63882) beads twice and eluted in 20 μL 10 Tris buffer, 8 μL of the eluents was taken to be barcoded in a 20 μL reaction in 6 cycles. The barcoded libraries were cleaned with AMPure beads twice and loaded onto MiSeq (Illumina, Cat. No. MS-102-3001) for sequencing 80 bases for Read1 and 70 bases for Read2, after quantitation following the manufacturer’s protocol^{14 (link)}.