variety: PUSA 372 tissue: Leaf age: 25-Days after sowing
Treatment protocol
The leaves of 25 days after sowing (DAS) old chickpea plants were spray-inoculated with A. brassicae spore suspension at the concentration of 5 X 10^6, containing the 0.05% Tween-20 (Cat # MB067, Himedia Laboratories, Mumbai). The spray was performed till the whole plant is fully drenched with the spore suspension. The Control plants for chickpea were spray inoculated with sterile water containing 0.05% Tween-20. Leaf samples were harvested at 24 hpi and 48 hpi after pathogen infection.
Growth protocol
The seeds were sown in soil mixture of agropeat (Prakruthi Agro Tech, Karnataka, India): vermiculite (Keltech Energies Ltd, Maharashtra, India) mix (3:1 volume/volume, pre-weighed. Plants were grown in short-day conditions with 8 h of light, 16 h of dark, 200 µE m-2s-1 light intensity, 75% humidity, and 21 °C constant temperature in growth chamber (PGR15, Conviron, Winnipeg, Canada). The bottom irrigation was done by water and Hogland solution (Cat # TS1094, Himedia Laboratories, Mumbai, India) thrice a week till the start of treatment.
Extracted molecule
total RNA
Extraction protocol
The total RNA for the samples was isolated by using TriZol reagent (Cat# 15596026 Invitrogen, Carlsbad, CA, USA) following manufacturers’ protocol. Then, RNA was cleaned by using RNeasy minikit (Cat#74104, Qiagen, Valencia, CA, USA) and further quantified by using NanoDrop ND-1000 spectrophotometer (Thermo Scientific, MA, USA). The RNA quality was examined by the Bioanalyzer (Agilent, Palo Alto, CA, USA).
Label
Cy3
Label protocol
The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). 500ng each of total RNA were reverse transcribed at 40°C using oligo dT primer tagged to a T7 polymerase promoter and converted to double stranded cDNA. Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up using Qiagen RNeasy columns (Qiagen, Cat No: 74106) and quality assessed for yields and specific activity using the Nanodrop ND-1000.
Hybridization protocol
600ng of labeled cRNA sample were fragmented at 60ºC and hybridized on to a Genotypic designed Chickpea GXP_8X60K (AMADID: 037094). Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit (Agilent Technologies, In situ Hybridization kit, Part Number 5190-0404). Hybridization was carried out in Agilent’s Surehyb Chambers at 65°C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Agilent Technologies, Part Number 5188-5327) and scanned using the Agilent Microarray Scanner (AgilentTechnologies, Part Number G2600D).
Scan protocol
Agilent Microarray Scanner (Agilent Technologies, Part Number G2600D).
Description
Leaf tissue infected with Alternaria brassicae, sample of 24 hpi, biological replicate 1
Data processing
Images were quantified using Feature Extraction Software ( Agilent). Feature extracted raw data was analyzed using GeneSpring GX software from Agilent. Normalization of the data was done in GeneSpring GX using the quantile normalization method . Gene based analysis was performed.
