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Single cells and pool-and-split cDNA libraries were constructed as published previously(Tang et al., 2010). In brief, individual cells are seeded into lysate buffer by mouth pipette, and reverse transcription reacted directly on the whole cell lysate. We then applied exonuclease I (New England Biolabs) to remove free primers. Next, a poly (A) tail was added to the 3’ end of the first-strand cDNAs using terminal deoxynucleotidyl transferase (Invitrogen). Single-cell cDNAs were then amplified by 33 cycles of PCR. The resulting 100–200 ng of amplified cDNAs were used to construct a sequencing library. Final cDNA libraries (200–2,000 ng depending on the amount of input material) were checked for known marker gene OsRBC (Os12g17600) using qPCR, and after passing quality control, libraries were fragmented with a Covaris S2 system. After fragmentation, we constructed cDNA library using the NEBNext Ultra DNA library Prep Kit for Illumina according to the manufacturer’s instructions. Samples were sequenced using Illumina HiSeq 2000 to obtain > 20 million single-end 100-bp reads per sample.
Institute of Genetics and Developmental Biology
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