We have developed a new method for the generation of cDNA from total RNA or from single cells, called Smart-Seq. Briefly, polyA+ RNA was reverse transcribed through tailed oligo-dT priming directly in total RNA or a whole cell lysate using Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT). Once the reverse transcription reaction reaches the 5′ end of an RNA molecule, the terminal transferase activity of MMLV adds a few non-templated nucleotides to the 3′ end of the cDNA. The carefully designed SMARTer II A oligo then base-pairs with these additional nucleotides, creating an extended template. The reverse transcriptase then switches templates and continues transcribing to the end of the oligonucleotide. The resulting full-length cDNA contains the complete 5′ end of the mRNA, as well as an anchor sequence that serves as a universal priming site for second strand synthesis. The cDNA is then amplified using 12 cycles for 1 ng of total RNA, 15 cycles for 100 pg of total RNA, and 18 cycles for 10 pg total RNA or from single cells. The exact number of cycles for each dilution replicate or single-cell is detailed in Supplementary Table 1. The Smart-Seq cDNA generation and amplification methods developed for this manuscript have recently become available in a kit marketed by Clontech called the “SMARTer Ultra Low RNA Kit for Illumina sequencing”. Although all the libraries in this manuscript were generated before the kit became commercially available, our protocol is reflected in the detailed instructions for generating cDNA from few cells or 100 pg–10 ng of total RNA that is now included in the manual for this kit. For single cell applications, each cell (or control RNA) was added in max 1 λ of media to 4 λ of hypotonic lysis buffer consisting of 0.2% Triton X-100 and 2 U/μl of ribonuclease (RNase) inhibitors (Clontech, 2313B) in RNase free water. The deposition of an intact cell in the hypotonic lysis buffer leads to immediate lysis and stabilization of the RNA through RNase inhibitors. Then, poly(A)+ RNA was reverse-transcribed through tailed oligo(dT) priming using the CDS primer (5′‐AAGCAGTGGTATCAACGCAGAGTACT(30)VN‐3′, where V represents A, C or G) directly in total RNA or a whole cell lysate using Moloney murine leukemia virus reverse transcriptase (MMLV RT).