title: GSM3557577: Root_minusSalt_plusDMS_rep3; Arabidopsis thaliana; OTHER
library_construction_protocol: Total RNA for the 24 individual biological samples was obtained in a chemical fume hood using the NucleoSpin RNA Plant kit. For each sample, 300 µg total RNA comprised the starting material for two rounds of poly(A) selection using the Poly(A)Purist MAG Kit, in order to obtain high purity mRNA for library construction. Reverse transcription was performed using SuperScript III First-Strand Synthesis System kit using the same RT primer as previously used (1): 5'CAGACGTGTGCTCTTCCGATCNNNNNN3' which is a fusion of a random hexamer and an Illumina TruSeq Adapter. The first-strand cDNA was size-selected above 52 nt on 8M urea 10% polyacrylamide gels to remove excess RT primer and increase the ligation efficiency in the next step. After recovering cDNA using the crush-soak method, the cDNA was dissolved in 6 µL RNase-free water. Ligation was performed using T4 DNA ligase which ligated the 3'end of the cDNA to a low bias single-stranded DNA linker /5Phos/TGAAGAGCCTAGTCGCTGTTCANNNNNNCTGCCCATAGAG/3SpC3/ where the underlined sequence can forms a hairpin structure and the random hexamer can then hybridize to any cDNA fragment. Reagents were added into the cDNA solution as follows: 2 µL 10x buffer, 2 µL 5M betaine, 1 µL 100 µM linker DNA, 8 µL 50% PEG8000, 1 µL T4 DNA ligase (400U/µL). The ligation was performed at 16°C for 6 h and then 30°C for 6 h, and the enzyme was then deactivated at 65°C for 15 min. The ligation product was size selected above 90 nts on 8M urea 10% polyacrylamide gels to remove extra single-stranded linker DNA and a 67 nt ligation byproduct, consisting of one copy of the hexamer and one copy of the linker DNA. After recovery using the crush-soak method, the purified ligation product was dissolved in 10 µL RNase-free water. PCR amplification was performed using a primer specific to the single-stranded linker DNA and fused with an Illumina TruSeq Universal Adapters.
attributes.tag: GEO Accession
attributes.value: GSM3557577