title: Illumina Genome Analyzer II sequencing; Cap analysis of gene expression (CAGE) of polysome-associated RNA against total RNA from human HEK293 cell line
design: Cap analysis of gene expression (CAGE) of polysome-associated RNA against total RNA from human HEK293 cell line
library_construction_protocol: HEK293 cells were cultured in Dulbecco's Minimal Essential Medium (GIBCO, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum and penicillin-streptomycin. The cells were harvested at the exponential growth period. Total RNA was extracted by using TRIzol reagent (Invitrogen, Cat.No 15596-018) after 1×PBS washing. Polysome fraction was collected using the method described in Bor et al. (2006) with minor modification. Around 80% confluent cells were incubated with 50 µg/ml cycloheximide for 30min at 37oC. Scraping cells into a 1.5 ml Eppendoff tube with a cell lifter. The mono- and polysome fraction was extracted with 250μl of 2×RSB/RNasin and 250 µl of polysome extraction buffer. Totally, 550μl of the cytoplasmic extraction was loaded onto 10-50% sucrose gradient which was made by the BioComp gradient master. The sucrose gradient was centrifuged with the SW41Ti rotor buckets at 36,000 rpm for 2 h at 4oC. Both monosome and polysome fraction was collected with the BioComp piston gradient fractionator. Total RNA was isolated with TRIzol reagent. In brief, ～1×106 cells were complete lysed with 1ml TRIzol by pipetting. 200ul Choloroform was added and shaked vigoursly for 15 seconds, then centrifuged at 12000 rpm for 15 min at 4oC. The total RNA was precipitated from aqueous phase with 500μl isopropyl alcohol. The mono- and polysome associated RNA was also extracted with the same method. The 27bp-tagged deep sequencing library was constructed using the method described in Valen et al. (2009) with some modifications. First-strand cDNA synthesis with 30μg total RNA and 8μg of anchored-N20 primer（5'- TTCCAGATACAGCAGN20). In brief, the mixture of total RNA and RT primer was heated at 65oC for 5min, then put on ice. 30µl of 5× GC buffer (Finnzyme), 4µl of 10mM dNTPs, 20 µl of 4.9M Sorbitol, 10µl of saturated Trehalose (>75% w/v), 8µl of SuperScript II (200U/µl, Invitrogen Cat No 18064014) were then added. The tube was incubated in a thermocycler at 25oC for 10 min, 42oC for 60 min, 56oC for 5 min and held at 4oC until further processing. The capped RNA was selected using the cap-trapper method described in Valen et al. (2009). The 2nd strand was synthesized by ligating a N6 adaptor (N6-up: Bio-ACAGGTTCAGAGTTCTACAGTCCGACGATCTAGCAGCAGN6 / N6-down: Pi-CTGCTGCTAGATCGTCGGACTGTAGAACTCTGAACCTGT) to the 3'end of the ssDNA with T4 DNA ligase (NEB) at 16oC overnight. The primer (5' Bio-AGAGTTCTACAGTCCGACGA 3') extension was performed by added 10× ExTaq buffer (Takara) 5μl and 2.5mM dNTP (Takara) 2μl ExTaq polymerase 1μl (Takara). The mixture was incubated at 65oC for 5min, 68oC for 30min, 72oC for 10min. The dsDNA was digested with EcoP15I(NEB), 10 μl 10× NEbuffer 3, 10μl 10× ATP solution, 1 μl 100× BSA, and 4 μl EcoP15I (10,000 U/μl) (Matsumura et al 2012). The reaction mixture was incubated at 37°C for2 h. An 3' adaptor (3' adaptor-up :NNTCGTATGCCGTCTTCTGCTTG. 3'adaptor-down: CAAGCAGAAGACGGCATACGA) was ligated to the recovered EcoP15I fragments. The PCR primer1 (5′-CAAGCAGAAGACGGCATACGA -3′) and PCR primer2 (5'-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3′) were used to create a deep sequencing library which contained the first 27bp from the 5' ends of capped RNA.