University) and used as described [25]. A Smad 3 expression plasmid was a gift from Prof. Lan Huiyao. MyHC and Troponin luciferase reporter (MyHC-Luc and Troponin-Luc) were used as described [25]. miR-29-promoter luciferase reporter was created and used as described (Wang et al., 2008). Renilla luciferase reporter was obtained from Promega and used according to manufactory. RT-PCR and Real-time RT-PCR Total RNAs from cells were extracted using TRIzol reagent (Invitrogen). Expression of mature miRNAs was determined using the miRNA-specific Taqman microRNA assay kit (Applied Biosystem) on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystem). U6 was used for normalization. Expression of mRNA analysis was performed with SYBR Green Master Mix (Bio-Rad Laboratories) as described using GAPDH for normalization [25]. Immunoblotting, Immunostaining and Immunohistochemistry For Western blotting analyses, total cell extracts were prepared and used as previously described [36]. The following dilutions were used for each antibody: Myogenin (Santa Cruz Biotechnol- ogy; 1:2000), YY1 (Santa Cruz Biotechnology; 1:2000), Troponin (Sigma; 1:2000), MyHC (Sigma; 2,000), Smad 2 (cell signaling; 1:2000), Smad 3 (Abcam; 1:2000), Smad 7 (Santa Cruz; 1:2000), Collagen 1 (Novus Biologicals, 1:2000), alpha Smooth Actin (a- SMA, Millipore; 1:2000), a-Tubulin (Sigma; 1:5000), and GAPDH (Santa Cruz Biotechnology; 1:5000). Immunofluores- cence of cultured cells was performed using the following antibodies: MyHC (Sigma; 1:350), Collagen 1 (Novus Biologicals, 1:350), a-SMA (Millipore, 1:400), Vimentin (Santa Cruz; 1:350), MyoD (DAKO, 1:400). All fluorescent images were captured with a confocal laser scanning microscope (FV1000, Olympus, Japan). Fluorescence was detected with an Olympus microscope (FV1000, Olympus, Tokyo, Japan). All samples were imaged with the 206 or 406 objective lens. Pictures were captured in Kahlman frame giving an average of two scans using the Olympus microscope FV1000 and the accompanying software FV10-ASW (version 01.07.02.02, Olympus). ChIP assays ChIP assays were performed as recommended by the manufacturer (Upstate) using 5 mg of antibodies against YY1 (Santa Cruz Biotechnology), Ezh2 (Cell signaling), trimethyl- histone H3-K27 (Millipore), Smad 3 (Abcam), or isotype IgG (Santa Cruz Biotechnology) used as a negative control. Genomic DNA pellets were resuspended in 20 ml of water. qRT-PCR was performed with 1 ml of immunoprecipitated material with SYBR Green Master Mix (Bio-Rad Laboratories). Relative recruitment is calculated as the amount of amplified DNA normalized to input and relative to values obtained after normal IgG immunoprecip- itation, which were set as 1. Primers used are indicated in the supporting information files. Animal studies Smad 3+/+ (C57BL/6) and Smad 32/2 (Smad3ex8/ex8 mouse), Smad 7+/+ (CD-1 or ICR) and Smad 72/2 (Smad7_exI mutant mouse) mice were kind gifts from Prof. Lan Huiyao [38,39]. Mice were housed in the animal facilities of The Chinese University of Hong Kong (CUHK) under conventional conditions with constant temperature and humidity and fed a standard diet. Animal experimentation was approved by the CUHK Animal Ethics miR-29 in Myoblast Conversion to Myofibroblast Committee. Primary myoblasts were isolated from approximately one week old mice muscles by the described procedures (Rando and Blau, 1994). Briefly, total hind limb muscles (3 to 6 mice per group) were digested with type IV collagenase (Invitrogen, 5 mg/ ml) and dispase II (Invitrogen, 1.4 mg/ml) for 0.5 hr, and cell suspensions were further homogenized by pipetting before being filtered through 70 mM and 40 mM filters. The obtained cells were pre-plated on uncoated cell culture plates in F10 media (Invitrogen) to selectively enrich for myoblasts. After two rounds of pre-plating, the cell suspension was plated on Gelatin-coated plates (Iwaki) in F10 medium (Invitrogen) supplemented with 20% FBS and Basic Fibroblast Growth Factor (Invitrogen, 25 ng/ml). Primary myoblasts were used at passage 3–5 after isolation. For Cardiotoxin injection. Approximately five week old Smad7+/+ or Smad72/2 mice were injected with 60 ml of cardiotoxin (CTX) at 10 mg/ml into the tibialis anterior muscles. Muscles were harvested at designated times, and total RNAs were extracted for real-time RT-PCR analysis. Sequencing and base calling Preparation of transcription libraries for sequencing on the Illumina GA2x platform was carried out using the mRNA-Seq Sample Preparation Kit (Cat # RS-930-1001) according to the manufactur- er’s standard protocol. Briefly, purified RNA was fragmented via incubation for 5 min at 94uC with the Illumina-supplied fragmenta- tion buffer. The first strand of cDNA was next synthesized by reverse transcription using random oligo primers. Second-strand synthesis was conducted by incubation with RNase H and DNA polymerase I. The resulting double-stranded DNA fragments were subsequently end-repaired, and A-nucleotide overhangs were added by incubation with Taq Klenow lacking exonuclease activity. After the attachment of anchor sequences, fragments were PCR-amplified using Illumina- supplied primers and loaded onto the GA2x flow cell. DNA clusters were generated with an Illumina cluster station with Paired-End Cluster Generation Kit v2 (Illumina), followed by 5162 cycles of sequencing on the GA2x (Illumina) with Sequencing Kit v3 (Illumina). Genome Analyzer Sequencing Control Software (SCS) v2.5, which could perform real-time image analysis and base calling, was used to carry out the image processing and base calling during the chemistry and imaging cycles of a sequencing run. The default parameters within the data analysis software (SCS v2.5) from Illumina were used to filter poor-quality reads. In the default setting, a read would be removed if a chastity of less than 0.6 is observed on two or more bases among the first 25 bases. Read mapping to genome with splice-aware aligner Sequenced fragments were mapped to UCSC mouse reference genome mm9 using TopHat version 1.1.4. Cufflinks version 1.0.0 was then used to estimate transcript abundances of RNA-Seq experiments. Abundances were reported in FPKM (fragments per kilobase of transcript per million fragments mapped) which is conceptually analogous to the reads per kilobase per million reads mapped (RPKM) used for single end RNA-seq. Statistical analysis Statistical significance was assessed by the Student’s t-test. (*p,0.5; **p,0.01; ***p,0.001) Supporting Information Figure S1 RNA-seq reveals that miR-29 overexpression leads to transcriptome change in C2C12 cells. Total RNAs were isolated from NC or miR-29 expressing C2C12 cells and subjected to high throughput mRNA sequencing (mRNA-seq). PLoS ONE | www.plosone.org 12 March 2012 | Volume 7 | Issue 3 | e33766