10 protocols
ID_REF = VALUE = signal intensity using rma function in affy package
The raw image files were processed by Bioconductor “Affy” and “vsn” package, the intensity of probes in arrays passed quality control were calculated and normalized for expression value.
GeneChip® Scanner 3000 7G 3000 (Affymetrix.Inc)
Hybridization, wash and Stain Kit(Affymetrix.Inc)
WT Terminal Labeling Kit (Affymetrix.Inc)
The reads generated by Illumina standard pipeline (CASAVA version 1.8) were mapped to mouse reference genome (mm9/NCBI37) using Bowtie (v0.12.7) software allowing the max mismatch 3nt. Only unique mapped reads were extracted for the following analysis. The aligned reads were further feed to MACS (v1.4.1) software to identify the DNA modification enriched regions (peaks). The MACS parameters were referred to the literature (default parameters with addition of --nolambda --nomodel -g mm) To adjust the library size variance and facilitate the data exhibition, the total 30 million mapped reads were randomly selected from each sample. Genome_build: mouse reference genome (mm9/NCBI37) Supplementary_files_format_and_content: Bed files were generated using MACS (v1.4.1), it contained the genomic coordinates of identified peaks
The cells were cultured until the appropriate stage. The culture medium were threw out and washed with PBS. The cells were centrifuge dissociated by 0.05% trypsin EDTA.The ESCs were added 2-folds-volume ESM and dissociated to single cells. The cell suspension were plated in new dished for 0.5hr(37℃) to clear away the feeder cells. The supernatant supension were further collected carefully and centrifuged for RNA extraction. The fibroblasts were centrifuged to collect the cells and treated with centrifuge centrifuged and treated with Trizol (Invirtrogen).
The iPS cells were cultured on mitomycin C treated MEFs in ES medium containing DMEM medium (Gibco Invitrogen, Carlsbad, CA) supplemented with 15% (v/v) fetal bovine serum (FBS), 1 mM L-glutamine, 0.1 mM mercaptoethanol, 1% nonessential amino acid stock, and 1000 U/ml LIF. The TSKM 2nd induction cells were deriven from TSKM iPS mice and cultured with Feeder Medium for 3 passages. On passages 4 the culture were changed to ES Medium with doxycycline for 3 days.
nucleic acid library construction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.(Invirtrogen)
nucleic acid library construction protocol
In brief, the sonicated genomic DNA was treated using NEBNext master Mix to repair the ends of DNA fragments and ligate the paired-end sequencing specific adaptors (Illumina), the DNA product was then immunoprecipitated and purified. Here we used 500ng ligation product for each IP reaction. Fragments were amplified with 14–16 cycles using adaptor specific primers (Illumina). The final product (300-500bp) was gel-purified (Qiagen) before cluster generation and sequencing.