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.
