CNhi10692_ACC.experiment
The cDNA synthesis is performed with RNA and N6+3 primer (5'-Pi-TCTNNNNNN-3') with SuperScript III RNase H- Reverse Transcriptase (Life Technologies) in the presence of 0.14M trehalose and 0.65M sorbitol. RNA/cDNA hybrid is purified with Agencourt RNAClean XP (BECKMAN). To elute the RNA/cDNA hybrid, the beads are resuspended with 37C pre-heated H2O and leaved at 37C for 5minutes. Diol residue is oxidized using 250mM NaIO4 on ice avoiding light. Capped RNA is biotinylated using 10mM Biotin dissolved in DMSO. After treated by RNaseOne, cDNA which is hybrid with biotinylated Capped RNA is selected by cap-trapper method. Binding condition is under 3.15M NaCl of Wash buffer 1 containing 0.1% Tween20. 15 ul of Dynabeads M-270 Streptavidin beads (Life Technologies) are used after coating with 3.75 ug of tRNA for 30 min. cDNA is released from beads with 65 ul of RNaseONE buffer at 95C for 5 min followed by a treatment with RNaseONE and RNaseH. The released cDNA is purified with Agencourt XP (BECKMAN) and eluted with 42 ul of 37C pre-heated H2O. The cDNA is treated with RNaseONE and purified with Agencourt XP again. On the other hand, quality of cDNA is checked using a house keeping gene by qRT-PCR. And quantification of cDNA is measured by Oligreen assay, A sample-specific 5'linker, containing a recognition site for a barcode sequence (3 bp), is ligated to the single-strand cDNA, which is concentrated with SpeedVac, with Mighty Mix (TAKARA). After ligation, the cDNA is purified with Agencourt AMPure XP (BECKMAN) twice to eliminate the unligated linkers followed by concentration with SpeedVac. A 3'linker  is ligated to the single-strand cDNA with Mighty Mix (TAKARA). After ligation, the cDNA is purified with Agencourt AMPure XP (BECKMAN) followed by SAP(Affymetrics) and USER (NEB) treatment to restrict the upper strand of 3'linker. The cDNA is purified with Agencourt AMPure XP (BECKMAN) and concentrated with SpeedVac. During purification and concentration, cDNAs are mixed into one tube. In this protocol, the number of samples able to be mixed is upto 24. The 2nd strand synthesis is performed by Deep Vent (exo-) DNA Polymerase (NEB) with nAnT-iCAGE 2nd primer, 5'- AATGATACGGCGACCACCGAGATCTACACTCTT TCCCTACACGACGCTCTTCCGATCT-3'. The resulting double-stranded cDNA is treated with ExonucleaseI (NEB) to eliminate extra primers followed by purification with Agencourt AMPure XP (BECKMAN) twice, 1.8x and 1.4x, and concentration with SpeedVac. Quantification of the final product is measured by Picogreen (Life Technologies) assay. And quality of the final product is checked by Agilent Bioanalyzer with High Sensitivity DNA kit. The final product is denatured with 2M NaOH and neutralized with 1M Tris-HCl (pH7.0) followed by dilution with Hybridization buffer HT1(Illumina). All of the diluted solution should be loaded on a flow cell.