High sensitivity dna chips for bioanalyzer

Whole transcriptome amplification of the mRNA was done using the Smart-seq protocol (Ramsköld et al., 2012 (link)). Briefly, Agencourt RNAClean beads were used to isolate the mRNA, followed by reverse transcription reaction using SMARTScribe (Clontech) enzyme. cDNA was isolated using Agencourt AMPure XP beads, followed by PCR amplification using the Advantage2 kit (Clontech). After another wash with the AMPure beads, DNA quality and concentration were measured on randomly selected wells using High Sensitivity DNA Chips for Bioanalyzer (Agilent technologies). This information was used to estimate DNA concentrations in the other wells, and to eliminate empty wells. Cells were labeled and sequencing libraries were prepared using Nextera XT DNA library preparation kit (Illumina), after wells were diluted in TE according to their estimated concentration. After all wells from a given plate were pooled and washed using AMPure XP beads, samples were sequenced using Rapid flowcell on an Illumina HiSeq2500 sequencer.

Representative cDNA from single cells across three C1 runs and Smart-Seq2 (on plates) were assessed using High Sensitivity DNA chips for Bioanalyzer (5067-4626 and 5067-4627; Agilent Technologies). Single cell cDNA from SMARTer3 (link),28 (link),29 (link),31 (link),32 (link) and Smart-Seq2 C1 IFCs and Smart-seq2 (on plates) was tagmented and pooled to make libraries using Illumina Nextera XT DNA sample preparation kit (Illumina; FC-131-1096) with 96 dual barcoded indices (Illumina; FC-131-1002). The library clean-up and sample pooling was performed using AMPure XP beads (Agencourt Biosciences; A63880). All protocols are described in the Fluidigm protocol (100-5950), Fluidigm Script Hub and Smart-seq2 protocol40 (link). The STRT-Seq libraries were made and sequenced at Karolinska Institutet as previously described9 (link),20 (link). The Single cell libraries from SMARTer and Smart-Seq2 C1 IFCs and Smart-seq2 (on plates) was sequenced across 1 lane of HiSeq V4 (Illumina) using 75bp/125bp paired-end sequencing.