Single-cell RNA-seq of tdTomato+ cells

Lysis plates were prepared by dispensing 4 μl lysis buffer as described in Schaum et al. (2018) (link). After dissociation, single tdTomato⁺ cells were sorted in 96-well plates using SH800S (Sony). Immediately after sorting, plates were sealed with a pre-labelled aluminum seal, centrifuged, and flash frozen on dry ice. cDNA synthesis and library preparation were performed using the Smart-seq2 protocol (Picelli et al., 2014 (link)). Wells of each library plate were pooled using a Mosquito liquid handler (TTP Labtech). Pooling was followed by two purifications using 0.7x AMPure beads (Fisher, A63881). Library quality was assessed using capillary electrophoresis on a Fragment Analyzer (AATI), and libraries were quantified by qPCR (Kapa Biosystems, KK4923) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Libraries were sequenced on the NextSeq 500 Sequencing System (Illumina) using 2 × 75 bp paired-end reads and 2 × 8 bp index reads.