GSM5088614: sft_dst_Single_Meristem_Transcriptomes; Solanum lycopersicum; RNA-Seq
RNA capture from shoot apical meristems for SMT:  The next steps for purification of RNA were conducted consecutively on batches of 24-96 meristems at each time, and typically within 1-2 hours: First, the samples were homogenized by putting the tubes (containing the glass bead) to bead beater instrument (Retsch, Mixer Mill MM 400) for 2 minutes. We then spin-downed the tubes and added 50ul of Lysis/Binding buffer (Dyanbeads, mRNA DIRECT Purification Kit) before applying another round of 2 minutes in bead beater. The homogenized samples were then kept on ice and transferred to 96-well plates before further RNA purification. In the 96-well plates, we captured RNA by adding 15uL of poly-dT beads (Dyanbeads, mRNA DIRECT Purification Kit) to each sample, which followed by washes and elution in 16-30uL of TRIS-HCl pH 7.5 Preparaion of bulk cDNA library by MARS-seq: 1ul purified RNA was transferred either manually, by an E1 Clip tip equalizer pipette (Thermo Fisher), or by a custom robotized dispensing protocol (Agilent, Automated liquid-handling platform, Bravo model with 96ST head)  into barcoded lysis solution (4nM of RT-barcodes) in 384-well MARS-seq plates, and by assigning 12-24 wells to each meriste to serve as technical replicates. During cDNA preparation, only deviation from the original MARS-seq scRNA protocol describied by Jaitin et al 2014 was extending the first evaportaion step at 95 degree from 3 to 4 minutes, in order to compensate for the additional volume. UMIs obtained from the 12-24 libraries originated from same meristem (technical replicates) were summed prior to downstream analysis of SMT data (see Meir, Aviezer et al. for graphical overview of the protocol).