RNA sequencing library construction for Illumina GA II
Fragment 400ng of amplified mRNA to 10‐200nt using 10x RNA fragmentation buffer ( Ambion ) .
Purify using regular ethanol precipitation method with 0.35μl of GlycoBlue ( Ambion ) .
Dephosphorylate the 3’ end the RNA samples using 10x Antarctic Phosphatase Buffer and 0.5 μl Antarctic Phosphatase ( NEB ) at 37 °C for 20 minutes .
Heat inactivate at 75°C for 10 minutes .
Phosphorylate the 5’ end of RNA samples was using 10x T4 DNA ligase buffer ( it has 1mM ATP final ) and T4 PNK ( NEB ) at 37 °C for 30 minutes .
Purify the RNAs in the reactions using ammonium acetate and ethanol precipitation with 2μl of GlycoBlue ( Ambion ) .
Ligate the RNA samples at 37 °C for one hour to 3’ linker ( 5’‐ / 5rApp / CTG TAG GCA CCA TCA AT / 3ddC / ‐3’ ) ( synthesized by IDT ) using : T4 RNA ligase 1 ( NEB ) , 5X ATP‐free T4 RNA ligase buffer ( 16.5 mM DTT , 41.5 % glycerol , 250 mM HEPES‐KOH , pH8.3 , 50 mM MgCl2 , 50 μg / ml acetylated BSA ) , and 10 % DMSO .
Purify the RNAs in the reactions using ammonium acetate and ethanol precipitation with 2μl of GlycoBlue ( Ambion ) .
Run the RNA samples on 6 % TBE‐Urea PAGE Gel ( Invitrogen ) .
Cut 100‐200nt bands and elute overnight with 400μl stop solution ( 1M ammonium acetate and 10mM EDTA ) at 4°C .
Purify the RNAs in the supernatant using regular ethanol precipitation method with 2μl of GlycoBlue ( Ambion ) .
Ligate the RNA samples at 37°C for 1 hour to 5’ linker ( with bar code ) using : T4 RNA ligase 1 ( NEB ) , 10x T4 RNA ligase 1 buffer ( NEB ) , and 10 % DMSO .
Purify the RNAs by ammonium acetate and ethanol precipitation and gel purification as described in steps 8 - 11 .
Reverse transcribe cDNA of the RNA samples using SuperScript III ( Invitrogen ) following manufacture’s protocol .
The primer sequence used for reverse transcription is 5’‐ATT GAT GGT GCC TAC AG‐3’ .
Amplify the cDNA samples using Taq ( NEB ) following manufacture’s protocol .
Purify the PCR products ( 200‐300nt ) using Qiagen PCR purification kit .
Dilute the purified PCR samples to 10nM and sequence using Illumina GA II sequencing system .
