Neutralize tagment buffer

cDNA synthesis was performed using the plate-based SmartSeq2 protocol^{41 (link)}, with 20 cycles of amplification during the PCR step. Amplified cDNA was purified twice with 0.7x AMPure beads (Fisher A63881). cDNA quality and concentration were then assessed by capillary electrophoresis on a Fragment analyzer (AATI) before sequencing library preparation. Illumina sequencing libraries for cDNA from single cells were prepared as previously described^{41 (link)}. Briefly, cDNA libraries were prepared using the Nextera XT Library Sample Preparation kit (Illumina, FC-131–1096). Nextera tagmentation DNA buffer (Illumina) and Tn5 enzyme (Illumina) were added, and the sample was incubated at 55°C for 10 minutes. The reaction was neutralized by adding “Neutralize Tagment Buffer” (Illumina) and centrifuging at room temperature at 3,220 × g for 5 minutes. Samples were then indexed via PCR by adding i5 indexing primer, i7 indexing primer, and Nextera NPM mix (Illumina). Following cDNA sequencing library preparation, wells of each library plate were pooled using a Mosquito liquid handler (TTP Labtech), then purified twice using 0.7x AMPure beads. Libraries were sequenced on a NextSeq 500 (Illumina) using 75bp paired-end sequencing.

cDNA synthesis was performed using the Smart-seq2 protocol [1,2]. Illumina sequencing libraries were prepared according to the protocol in the Nextera XT Library Sample Preparation kit (Illumina, FC-131–1096). Each well was mixed with 0.8 μl Nextera tagmentation DNA buffer (Illumina) and 0.4 μl Tn5 enzyme (Illumina), then incubated at 55°C for 10 min. The reaction was stopped by adding 0.4 μl ‘Neutralize Tagment Buffer’ (Illumina) and spinning at room temperature in a centrifuge at 3220 × g for 5 min. Indexing PCR reactions were performed by adding 0.4 μl of 5 μM i5 indexing primer, 0.4 μl of 5 μM i7 indexing primer, and 1.2 μl of Nextera NPM mix (Illumina). PCR amplification was carried out on a ProFlex 2 × 384 thermal cycler using the following program: 1. 72°C for 3 min, 2. 95°C for 30 s, 3. 12 cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 1 min, and 4. 72°C for 5 min.