Drop-Seq: NGS preparation of mRNA and SBOs

For each experiment, samples of various conditions were pooled together. The pooled cells were passed through a 40-μm filter and diluted at a final combined concentration of 100 to 400 cells/μl according to Drop-Seq protocol instructions (4). Droplets were generated and processed as previously described. Droplets were collected, and the recovered beads were processed for immediate reverse transcription, followed by exonuclease I treatment. The resulting complementary DNA (cDNA) was divided into appropriate number of tubes, amplified using the KAPA HiFi HotStart PCR Kit (Kapa Biosystems Inc., Switzerland). cDNA amplification was performed in 50 μl of polymerase chain reaction (PCR), which included 4 μl of 10 μM SMART PCR primer, 25 μl of KAPA HiFi DNA polymerase, and up to 21 μl of nuclease-free water. Then, PCR was performed using the following protocol: 3 min at 95°C; four cycles of 20 s at 98°C, 45 s at 65°C, 3 min at 72°C; nine cycles of 20 s at 98°C, 20 s at 67°C, 3 min s at 72°C; 5 min at 72°C. The PCR products were purified twice using 0.6× AMPure (Beckman Coulter, USA) beads according to the manufacturer’s instructions. To obtain reverse-transcribed SBOs that are much shorter than cDNA, the first supernatant from AMPure purification step was further purified adding 1.4× homemade AMPure beads [using Sera-Mag SpeedBeads (Thermo Scientific, USA), hereafter Serapure beads (21)]. The cDNA products were fragmented and further amplified using the Nextera XT DNA Library Preparation Kit (Illumina, USA).

The SBO library preparation was performed using a two-step PCR protocol. One nanogram of the SBO cDNA product was loaded into 20 μl of the first adaptor PCR, which included 1 μl of 10 μM forward and reverse primers, 10 μl of KAPA HiFi DNA polymerase, and up to 8 μl of nuclease-free water. PCR was performed using the following protocol: 3 min at 95°C; eight cycles of 20 s at 95°C, 20 s at 64°C, 20 s at 72°C; 5 min at 72°C using the following primers: SMART+AC; P7-SBO hybrid. After 1.8× Serapure bead purification, 8 μl of the first PCR product was loaded into 20 μl of the second index PCR, which included 1 μl of 10 μM forward and reverse primers, and 10 μl of KAPA HiFi DNA polymerase. PCR was performed using the following protocol: 3 min at 95°C; six cycles of 20 s at 95°C, 20 s at 60°C, 20 s at 72°C; 5 min at 72°C using the following primers: New-P5-SMART PCR hybrid; Nextera index oligo. The second PCR product was purified using 1.2× Serapure beads. All primers were prepared by IDT. Sequencing was performed on an Illumina NextSeq 500 system using a NextSeq 500/550 High Output v2 kit (75 cycles) (Illumina, USA). The sequences of primers were provided in table S1. The sequencing depth and number of cells of each experiment are provided in fig. S9.