Single-cell cDNA profiling and library preparation

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.

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Svensson V., Natarajan K.N., Ly L.H., Miragaia R.J., Labalette C., Macaulay I.C., Cvejic A, & Teichmann S.A. (2017). Power Analysis of Single Cell RNA-Sequencing Experiments. Nature methods, 14(4), 381-387.

Publication 2017

High Sensitivity DNA chips for Bioanalyzer Nextera XT DNA sample preparation kit 96 dual barcoded indices HiSeq V4

Cdna Dna chips Fc 131 Library Sensitivity

Corresponding Organization : European Bioinformatics Institute

Other organizations : Wellcome Sanger Institute

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Variable analysis

independent variables

Single cell cDNA from SMARTer3, Smart-Seq2 (on plates), and Smart-Seq2 C1 IFCs

dependent variables

Library clean-up and sample pooling using AMPure XP beads
Sequencing of single cell libraries across 1 lane of HiSeq V4 using 75bp/125bp paired-end sequencing

control variables

Protocols described in Fluidigm protocol (100-5950), Fluidigm Script Hub and Smart-seq2 protocol40 (link)
STRT-Seq libraries made and sequenced at Karolinska Institutet as previously described9 (link),20 (link)

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