Illumina HiSeq 2500 sequencing; RNA-seq of Barrelette neurons and progenitors during mouse brain development
Cells were dissociated by papain, filtered and kept on ice until FACS sorting. Sorted cells were collected into ice cold PBS 1X, and immediately pelleted by centrifugation (500 rcf, 5 minutes, 4°C).  Total RNA was extracted by NORGEN Single Cell RNA Purification Kit (NORGEN, 51800) with genomic DNA digestion using RNase-Free DNase Ⅰ Kit (NORGEN, 25710) according to manufacturer's protocol.  Sequencing of poly A+ mRNA was done by Smart-seq2 protocol. 1ng of total RNA was used as an input. Reverse transcription was conducted using 100U SuperScript Ⅱ reverse transcriptase (Thermo Fisher, 18064014), 10U RNase inhibitor (Clontech, 2313A), 1X Superscript Ⅱ first-strand buffer, 5mM DTT (contained in SuperScript Ⅱ reverse transcriptase), 1M Betaine (Sigma, B0300-1VL), 6mM MgCl2, 1μM template-switching oligos (TSOs) (exiqon), 1μM oligo-dT primer (Mycrosynth) and 1mM dNTP mix (Thermo Fisher, R0191) in total volume 10μl. Then PCR pre-amplification was done using 1X KAPA HiFi HotStart Ready Mix (KAPA Biosystems, KK2602), 0.1μM IS PCR primers (Mycrosynth) in total volume 25μl using 13 cycles of PCR. DNA was purified with SPRI AMPure XP beads (Beckman, sample to beads ratio 0.8:1) and eluted in 20μl 10mM Tris HCl pH8. Tagmentation reaction was done by Illumina Nextera XT DNA Library Prep Kit (Illumina, FC-131-1024) in total volume 5μl using 0.2ng DNA as an input, then amplification of adapter-ligated fragment was done using Illumina XT kits in a total volume 12.5μl with 12 cycles of PCR. Library was purified by SPRI AMPure XP beads (sample to beads ratio 0.6:1) and eluted in 12μl 10mM Tris HCl pH8.