Fig. 1

Experimental design and RNA sequencing statistics by experimental group. a Dilution experiment summary. See Methods for detailed information. b Single cell amplification methods used. Protocols involve two key steps: conversion of RNA (blue) to cDNA (green), and amplification of cDNA. aRNA targeted poly-adenylated mRNA by using an oligo-dT T7 primer for initial cDNA synthesis. After generating double-stranded cDNA, molecules were amplified using in vitro transcription with T7 polymerase. This amplification procedure was designed to minimize exponential expansion of errors. cDNA generation and amplification were repeated two additional times before library preparation. SmartSeq Plus targeted total RNA using a mixture of poly-T and randomized primers for initial cDNA synthesis. Full-length transcripts were captured through the template-switching capacity of reverse transcriptase. Double stranded cDNA molecules were amplified using 18 rounds of PCR. All cDNA and amplification reactions were performed on a 96-well Fluidigm C1 chip, intended to reduce experimental variation by performing reactions in small volume. NuGen targeted total RNA through use of proprietary primers for initial cDNA synthesis. Second strand cDNA synthesis was generated using an RNA primer, which was subsequently degraded from the second strand cDNA copy, resulting in linear amplification by DNA replication. This method was designed to minimize exponential amplification of error. c Sample sizes and RNA sequencing statistics by experimental group. Includes color key used in all figures. For analysis based on combined HBR and UHR dilution replicates, solid colors were used. Abbreviations: Human Brain Reference (HBR), Universal Human Reference (UHR), University of Pennsylvania (Penn), University of California San Diego (UCSD), University of Southern California (USC), picogram (pg.), base pair (bp.), contamination (contamin.), average (Ave.), standard deviation (Sd.), amplification (amp.)