Single Nuclei Sequencing via Smart-seq2 Protocol

Smart-seq2 protocol for single nuclei sequencing was performed as described in^{60 (link)} with several modifications^{61 (link)}. In brief, individual nuclei were lysed, RNA lysate was reverse transcribed with 100U of Superscript III (Takara) with the addition of 10U of RNaseIn, 5 mM DTT, 1 M betaine (Sigma), and 6 mM MgCl2 (Invitrogen). Reverse transcription reaction was paused after 90 min at 42 °C for adding TSO oligos at the final concentration of 0.5 uM and continued for additional 12 min. The cDNA was amplified using KAPA HotStart HIFI 2 × ReadyMix (Kapa Biosystems) and 10 uM ISPCR primer for 24 cycles (98 °C – 3 min; 10 cycles: 98 °C – 20 s, 60 °C – 1 min, 72 °C – 6 min; 7 cycles: 98 °C – 20 s, 64 °C – 1 min, 72 °C – 6 min; 7 cycles: 98 °C – 20 s, 67 °C – 1 min, 72 °C – 6 min; 72 °C – 10 min; 4 °C – hold). Amplicons were purified by means of Ampure XP beads (Beckman Coulter, USA) at a sample:bead ratio of 1:1, their concentration was assessed using Qubit dsDNA HS Assay Kit (Invitrogen), while the amplicon quality was estimated by cDNA length on high-sensitivity DNA chip (Agilent) on Agilent 2100 Bioanalyzer. Oligo-dT, TSO and IS PCR primers (sequences are listed in Table S2) were ordered from Exicon (http://www.exiqon.com/). All primer sequences are listed in the Table S1.