title: Agrobacterium tumefaciens C58 whole transcriptome sequencing
design: cDNA library preparation. cDNA libraries were prepared and analyzed at Illumina (San Diego, CA). The detailed protocol describes the steps for total RNA fragmentation, adapter ligation, reverse transcription, PCR amplification, purification, cluster generation and sequencing, and can be found in Illumina TruSeq Small RNA SamplePrep Guide (#15004197).   Total RNA fragmentation: All purified total-RNA samples were started out with 100ng in total volume of 16µl.  Each sample was treated with 2µl of 5X fragmentation buffer (EPF#15016648, Illumina) and was incubated at 94°C for 4 minutes. Then the samples were cooled on ice.    PNK treatment: The sample was mixed with a 7µl master mix as following: 1µl of RNAseOUT (40U/µl) from Epicentre or RNAse Inhibitor (part#15003548, Illumina), 2µl of T4 Polynucleotide Kinase (PNK) (part#M0201S, NEB), 2µl of 10X PNK Buffer (part#M0201S, NEB), and 2µl of 10mM ATP (part#R109AT, Epicentre/Illumina). A 25µl reaction mixture was incubated at 37°C for 1 hour on a pre-heated thermal cycler. The fragmented total-RNA samples (small RNA) were purified with procedure 1 of the RNA clean & concentrator-5 (part#R1015, Zymo Research), and were then eluted with 6µl of RNase-free water.     3’ adapter ligation: First, 5µl of purified fragmented RNAs were ligated with 1µl RNA 3’ Adapter (RA3) (part# 15013207, Illumina); reactions were heated at 70°C for 2minutes, then immediately cooled on ice. Next, a master mix of 4µl was prepared as following before adding to the 6µl reaction: 2µl Ligation Buffer (HML) (part#15013206, Illumina), 1µl RNase Inhibitor (part#15003548, Illumina), and 1µl T4 RNA Ligase 2 Deletion Mutant (part# M0242S, NEB). The 10ul reaction was incubated on the pre-heated thermal cycler at 28°C for 1 hour. With the reaction tube remaining on the thermal cycler, 1µl Stop Solution (STP) (part#15016304, Illumina) was added to the reaction tube and mixed thoroughly by pipetting. Then the reaction mixture was incubated at 28°C for additional 15 minutes.   5’ adapter ligation:  1µl RNA 5’ Adapter (RA5) (part#15013205, Illumina) was added into the 11µl of the 3’ adapter ligation reaction mixture, and the sample was denatured at 70°C for 2 minutes and immediately cooled on ice. 1µl of 10mM ATP (part#15007432, Illumina) and 1µl of 10U T4 RNA ligase (part#1000587, Illumina) were added into the reaction to bring the final volume of 14µl that was incubated at 28°C for another hour and then placed on ice.  Reverse transcription: For first strand cDNA synthesis, 6 µl of the 14µl of 3’ and 5’ adapter ligated RNA samples was mixed with 1µl RNA RT Primer (RTP) (part#15013981, Illumina), denatured at 70°C for 2 minutes, and then immediately cooled on ice. 5.5µl of master mix containing 2µl 5X First Strand Buffer (part#18064-014, Invitrogen), 0.5µl 12.5 mM dNTP mix (dilute from 25mM dNTP mix, part #11318102, Illumina), 1µl 100 mM DTT (part#18064-014, Invitrogen), 1µl RNAse Inhibitor (part#15003548, Illumina), and 1µl SuperScript II Reverse Transcriptase (part#18064-014, Invitrogen) was added and incubated at 50°C for 1 hour on a pre-heated thermal cycler.   PCR amplification: A 50µl PCR reaction was set up by adding 8.5µl Ultra-Pure Water (part#1001913, Illumina), 25µl PCR Mix (PML) (part#15022681, Illumina), 2µl RNA PCR Primer (RP1) (part#15013198, Illumina), and 2µl RNA PCR Primer Index (RPI1) (part#15013181, Illumina) into the 12.5ul of the first strand cDNA reaction. PCR reaction was carried out in a thermal cycler with following profile: 98°C for 30sec followed by 11 cycles of 98°C for 10sec, 60°C for 30sec, 72°C for 15 sec, and a final extension at 72°C for 10 min. The PCR product was held at 4°C until purification.