title: Muscle tissue_CTRL_DMD_CAGEseq L1 and Alu profiling Nuclear_DMD_MT-1_9981_33
design: Library Construction:  The preparation of the nanoCAGE library was adapted from published protocol(Salimullah et al. 2009) and modified to work with Illumina Genome Analyzer IIx (GAIIx) 36 cycle Single Read sequencer.   The first-strand cDNAs were created using 100 µg Nuclear and cytoplasm RNA with 2 µl of 0.66 M D-threalose, 3.3 M D-sorbitol (Carninci et al. 1998), 100 µM template switching oligonucleotide (5’- TAGTCGAACTGAAGGTCTCCAGCArGrGrG), 10 µM random reverse-transcription primer with a random pentadecamer tail (5’- GTACCAGCAGTAGTCGAACTGAAGGTCTCCTCTN15). The mixed solution was reduced volume to 2 µl in a centrifugal evaporator at room temperature then heated for denaturizing at 65 °C for 10 min and transferred quickly on an ice-water mix. Reverse transcription was accomplished in a volume of 10 µl with the following components: 1.25 ×  first-strand buffer, 650 µM dNTPs, 1.3 mM DTT, 925 mM betain and 200 units SuperScript II, and the reaction was incubated at 22 °C for 10 min, 50 °C for 30 min, 75 °C for 15 min. Synthesized cDNAs were purified with Agencourt RNAClean XP kit following by the manufacturer’s instructions. The purified cDNAs were eluted to 40 µl water then synthesized the second strand cDNA and the resulting product by semisuppressive PCR (Plessy et al. 2010). The determination of the optimal PCR cycle number in pilot reactions was used quantitative PCR: 0.15 × purified cDNA, 1× SYBR Premix Ex Taq (TaKaRa), 100 mM semisuppressive forward primer (5’- TAGTCGAACTGAAGGTCTCCAGC) and 100 mM semisuppressive reverse primer (5’-TGACGTCGTCTAGTCGAACTGAAGGTCTCCGAACC) with a StepOne Real-Time PCR system. The thermal cycling program was: 5 min 95 °C and  40 × (15 s at 95 °C, 10 s at 65 °C, 2 min at 68 °C). After determination, PCR was performed a large-scale hot-start PCR with 3 µl of purified first-strand cDNA as template in a final volume of 50 µl using a reaction mixture containing 1.25 U TaKaRa Ex Taq HS (TaKaRa), 200 µM each dNTP mixture (TaKaRa), 1× Ex Taq buffer (TaKaRa), 100 mM semisuppressive forward primer and 100 mM semisuppressive reverse primer with the following thermal cycling program: 5 min 95 °C and n × (15 s at 95 °C, 10 s at 65 °C, 2 min at 68 °C). The amplified DNA was purified with Agencourt AMPure XP kit then measured the concentration by NanoDrop 1000.  To create adaptor sequences for Illumina GAIIx sequencer, semisuppressive PCR cDNA was amplified using 20 ng DNA in a final volume of 50 µl with a mixture containing 1.25 U Ex Taq HS (TaKaRa), 200 µM each dNTP mixture (TaKaRa), 1× Ex Taq buffer (TaKaRa), 200 mM forward primer (5’ – AATGATACGGCGACCACCGAGATCTACACTAGTCGAACTGAAGG) and 200 mM reverse primer (5’ – CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT) following thermal cycling program: 1 min 95 °C, 15 s at 95 °C, 10 s at 55 °C, 2 min at 68 °C and 6 × (15 s at 95 °C, 10 s at 65 °C, 2 min at 68 °C). The amplified DNA was purified with Agencourt AMPure XP kit then measured the concentration by Agilent High Sensitivity DNA kit and adjusted 16 pM final concentration (from 200 bp – 700 bp) on GAIIx. nanoCAGE tags were amprified with Cluster generation kit with modified sequence primer (5’ – TAGTCGAACTGAAGGTCTCCAGCA) following by Cluster generation protocol (Illumina) then  generated clusters were sequenced with 36 cycle Single Read sequencing kit by GAIIx sequencer.    Experimental Design:  We utilized deep nanoCAGE (Cap Analysis Gene Expression) sequencing (Carninci 2009), able to identify active transcription start sites (TSS)  at single-base-pair resolution, to survey the repetitive element transcriptome of human primary skeletal muscle cell progenitors undergoing differentiation. Separate nuclear and cytoplasmic RNA fractions of muscle primary cells from healthy (n=3 CTRL) and DMD-affected (n=3 DMD) donors of European origin were tracked throughout three stages of differentiation; proliferating myoblasts (MB), confluent myoblasts (MT-1) and day-7 myotubes (MT-7), producing our total set of data (N=36).