Smarter ultra low rna kit for sequencing hv

Library preparation was performed using 5 ng of total RNA. RNA integrity was assessed using an Agilent Bioanalyzer. cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing-HV (Clontech) per manufacturer’s instructions. cDNA was fragmented using a Covaris E210 sonicator using duty cycle 10, intensity 5, cycles/burst 200, time 180 seconds. cDNA was blunt-ended, had an ‘A’ base added to the 3′ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were amplified for 12 cycles using primers incorporating unique index tags. Replicate libraries from both cell types were pooled in equimolar ratios and sequenced on an Illumina HiSeq 2500 (single-end 50 bp reads) (Supplementary Table S1).

Sorted bipolar cell populations were resuspended in 500 µl TRIzol reagent (Invitrogen), and RNA was isolated according to manufacturer’s instructions. Prior to sequencing, RNA quality was analyzed using an Agilent Bioanalyzer. cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing-HV (Clontech) per manufacturer’s instructions. cDNA was fragmented using a Covaris E210 sonicator using duty cycle 10, intensity 5, cycles/burst 200, time 180 s. cDNA was blunt-ended, an ‘A’ base added to the 3′ ends, and Illumina sequencing adapters were ligated to the ends of the cDNAs. Ligated fragments were amplified for 12 cycles using primers incorporating unique index tags. Replicate libraries from each bipolar cell population were pooled in equimolar ratios and sequenced on an Illumina HiSeq 3000 (single-end 50 bp reads). For qPCR, RNA samples were treated with TURBO DNase (Invitrogen) and cDNA was synthesized with SuperScript IV (Invitrogen) and oligo(dT) primers according to manufacturer’s instructions. For Figure 1C, expression was normalized to the average of reference genes Gapdh, Sdha, Hprt, and Pgk. For Figure 1—figure supplement 2, expression was normalized to Gapdh alone. Primers for Grm6, Gnat1, Lhx1, Pax6, Rlbp1, Slc17a6, Vsx2, Grik1, Tacr3, Isl1, and Lrrtm1 are listed in Supplementary file 2.

RNA was extracted from single cells and processed for cDNA synthesis using the SMARTer Ultra Low RNA Kit for Illumina Sequencing-HV (Clontech Laboratories). Libraries were prepared using Clontech Low Input Library Prep Kit according to the manufacturer's instructions. An extended protocol can be found in the supplementary material Methods.