Single-Cell RNA-Seq Library Preparation

The eluted single-stranded library was stripped of RNA by adding 2 μl of RNase H (NEB M0297L), 4 μl of 10× RNase H buffer (NEB B0297S) and incubating for 30 min at 37 °C. The reaction was purified with a 1.8× solid phase reversible immobilization (SPRI), where the final eluate volume was 25 μl. To perform second-strand synthesis, we used a modified version^{42 (link)}, where we added 8 μl of 5× Maxima H- reverse transcription buffer, 4 μl 10 µM dNTPs, 2.5 μl of Klenow Fragment (3’ -> 5’ exo -, NEB M0212L), 5 μl 50% PEG 8000 and 1.5 μl 100 µM S^3 randomer (oBW140). The reaction was incubated at 37 °C for 60 min, cleaned with a 1.8× SPRI and eluted in 30 μl of nuclease-free water. The full length, double-stranded library was amplified using PCR by adding 30 μl of 2× Q5 High Fidelity master mix (NEB M0492L), 0.4 μl 100 µM oDS028 and 0.4 μl 100 µM oBW170. We amplified the library using the following protocol: 98 °C for 30 s, 14 cycles of 98 °C for 20 s, 65 °C for 30 s, 72 °C for 3 min. Following the first round of PCR, the reaction was cleaned twice, each time using a 1.2× SPRI reaction, and eluting in 40 μl. This was to ensure primer dimers were properly removed. The resulting samples were the gene expression (GEX) libraries.