RNAseq reads from shoot apical meristem samples in constant shade conditions
Tomato plants were grown in low R:FR conditions for the constant constant shade condition. Vegetative apices were harvested 19 days after planting. Shoot apical meristems were collected using Laser Capture Microdissection. Approximately 4 shoot apical meristems (SAMs) were microdissected per biological replicate. ~100,000μm2 of 10μm-thick sections were captured for SAM (containing the dome of the SAM and the incipient leaf, or P0). Library preparation for RNA-Seq was performed as described in Kumar et al., 2012) except for the following changes: For second strand synthesis, we added 10μl of cDNA (100-250ng), 0.5μl of random primers and 0.5μl of dNTP. Sample was mixed and heated at 80ºC for 2 min, 60ºC for 10 sec, 50ºC for 10 sec, 40ºC for 10sec, 30ºC for 10 sec and 4ºC for at least 2-5 min. We then added the following (on ice):  5ul of 10 X DNA polymerase I buffer, 31.5ul of H2O and 2.5 ul of DNA Polymerase I, followed by incubation at 16 ºC for 2.5 hours.  From this point onward the published RNA-Seq protocol (Kumar et al., 2012) was followed, except for the fragmentation time, which in this case was 20 min.