title: GSM2722734: Proplastid containg, no chlorophyll cells 1; Solanum lycopersicum; RNA-Seq
library_construction_protocol: Approximately 2 mm long SAM was dissected from seedlings was inserted into TissueTek@ OCT (Sakura Finetek) medium kept into 10x10x5 cm cryomoulds (Sakura Finetek) and freezed in Liq N2. Cryoblocks were cut in 10 um thick cryosections with cryostat (Leica CM3050 S.) 12-15 sections can be obtained from a single SAM. Sections were collected on 0.1mm Polyethylene Terephthalate (PET) or 1.0 mm Polyethylene Naphthalate (PEN) membrane slides. Different cell types were visualized based on their Chl fluorescence seen through DAPI filter (Ex 400; Em 460). Since the Chl flu is lost during ethanol fixation, flu images of some SAM section were taken and the adjacent sections were dissected based on these images. Sections were immediately fixed in 70%, dehydrated with 70%, 96% and 100%, 100%  ethanol, and dried in air for 2 min. LM was performed with a P.A.L.M.MicroBeam system mounted on AxioObserver microscope (Carl Zeiss). Cut areas were collected in 500ul adhesive caps containing 35 μl of RLT lysis buffer from RNeasy Micro RNA isolation kit (Qiagen), was put in cap. Cap was closed and put inverted for 30 min at room temp, then stored at -80 0C. RNA was isolated with Qiagen MicroRNA kit according to manufacturer’s instruction and quality was checked with Bioanalyzer 2100 (BA) with BA-PICO 6000 Chip (Agilent Technologies). RT reaction was performed with indexed primers containing from 5' to 3' end is: T7 promoter, Illumina 5' adaptor, Unique barcode, UMI and polyT sequence, to do first strand cDNA synthesis for all the RNA samples separately. External RNA controls Consortium (ERCC) mRNA spike-ins (Ambion, Life Technologies) were added to the samples during initial step of first strand cDNA synthesis. Different indexes/barcodes were used for different samples. Second strand cDNA synthesis was also performed separately on each sample. After second strand cDNA synthesis, samples containing the different barcodes were pooled and further processing was performed with this single sample. One round RNA amplification was carried out with Ambion/Life Technology (Invitrogen) MessageAmp™ II aRNA Amplification Kit (AM1751) as described by the manufacturer using 1/10 of the reaction volume as per Hashimshony et al., 2012, to get amplified RNA (aRNA).  Sample volume of aRNA was adjusted to 16μl, either by adding water or drying down in a speedvac, depending on RNA concentration. Reaction was end repaired by treating with phosphatase at 37 °C  for 30 min and with PNK at 37 °C  for 60 min as described in supplementary methods of Hashimshony et al., 2012 , and cleaned on RNeasy spin column. 3’ adapter was ligated using T4 ligase at 28 °C  for (60 +15) min. Reaction was stopped, reverse transcribed and PCR amplified with following program 30 seconds at 98°C, 18 cycles of: 10 seconds at 98°C, 30 seconds at 60°C, 30 seconds at 72°C and 10 minutes at 72°C. PCR product was cleaned with AMPure XP Beads (Agencourt AMPure XP). Quality of library was checked on Bioanalyzer (BA).