GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2722741

Query DataSets for GSM2722741

Status

Public on May 21, 2019

Title

Highly developed thylakoid and chlorophyll containg cells 2

Sample type

SRA

Source name

Leaf Primordium (LP)

Organism

Solanum lycopersicum

Characteristics

cell type: M82 SP+

Growth protocol

12-14 d old tomato (M82 SP+) seedlings were used as study material. Tomato seedlings were grown aseptically in magenta boxes at 200 mm m-2 s-1 light in long day conditions (11 h light, 13 h dark) at 21-23 0C, in half strength Nitch medium (Nitsch and Nitsch 1969).

Extracted molecule

total RNA

Extraction 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, 15 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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Tzach_DT_sample_0008

Data processing

Reads were demultiplexed for the different samples
Reads were aligned to the Solanum lycopersicum genome (Build 2.5) together with the Chloroplast sequence (NC_007898.3) using Bowtie2 (V 2.1.0) using local mode.
Reads with identical UMIs that are counted to a specific gene in the counting stage, are collapsed to a single read
Read counting to ITAG2.4 genes and to the Chloroplast annotations was done with HTseq-count (200 bp were added to the 3' end of each gene (not in the chloroplast genes due to short distances between genes) in order to ensure counting all reads per gene)
Differential expression analysis was performed with DESeq2 (1.4.0)
Genome_build: Solanum lycopersicum genome Build 2.5 & Chloroplast NC_007898.3
Supplementary_files_format_and_content: Excel file includes the DESeq2 normalized counts

Submission date

Jul 31, 2017

Last update date

May 23, 2019

Contact name

Gilgi Friedlander

Organization name

Weizmann Institute of Science

Street address

Hertzel st

City

Rehovot