Protocols

Myers Lab Inducible shRNA-K562 Protocol

K562-shX cells are made in an effort to validate TFBS data and ChIP-seq antibodies in Myers lab. K562 cells are transduced with lentiviral vector having Tet-inducible shRNA targeting a transcription factor gene. Cells with stable integration of shRNA constructs are selected using puromycin in growth media. Doxycycline is added to the growth media to induce the expression of shRNA and a red fluorescent protein marker. A successful shRNA cell line shows at least a 70% reduction in expression of the target transcription factor as measured by qPCR. For identification, we designated these cell lines as K562-shX, where X is the transcription factor targeted by shRNA and K562 denotes the parent cell line. For example, K562-shATF3 cells are K562 derived cells selected for stable integration of shRNA targeting the transcription factor ATF3 gene and showed at least a 70% reduction in the expression of ATF3 gene when measured by qPCR. Cells growing without doxycycline (uninduced) are used as a control to measure the change in expression of target transcription factor gene after induction of shRNA using doxycycline. To identify the potential downstream targets of the candidate transcription factor, analyze the mRNA expression profile of the uninduced and induced K562-shX using RNA-seq.

 

Myers Lab RRBS Protocol (Edited 06-2010)

Reduced Representation Bisulfite Sequencing (RRBS) is used to measure methylation status at more than 500,000 CpG dinucleotides in the genome. Genomic DNA is digested with the methyl-insensitive restriction enzyme Msp1, small fragments are purified by gel electrophoresis, and they are then used to construct an Illumina sequencing library that is treated with bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymidine. The sequenced fragments are aligned to a customized reference genome sequence and used to calculate the number of reads at each CpG assayed and the percentage of those reads that detect methylation.

 

Myers Lab ChIP-seq Protocol v041610

Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) is used to identify transcription factor binding sites in the genome. Transcription factors (TF) bound to genomic DNA are crosslinked to preserve these interactions, the DNA is fragmented and the DNA::TF complexes are immunoprecipitated (IP) using an antibody to the TF. The crosslinks are reversed and the DNA fragments are used to make and Illumina sequencing library that is then sequenced on the GAIIx. The sequenced fragments are compared to a background sequencing library with a peak-calling algorithm to identify DNA sequences enriched by IP that are likely bound by the transcription factor or a complex that includes the immunoprecipitated TF.

Myers Lab ChIP-seq Protocol v042211

Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) is used to identify transcription factor binding sites in the genome. This ChIP-seq protocol differs from v041610 in that it reduces the ChIP incubation times, uses the Bioruptor Twin (Diagenode) for chromatin sonication and Agencourt AMpure beads for size purification.