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Protocol publication

[…] ., ChIP-PCR of RNA pol II within the chromosome 3 and chromosome 7 regions was determined by 30 cycles of amplification of ChIP DNA using [α-32P] dCTP as tracer and primers listed in . Input reactions contain 1/100th fraction of chromatin DNA used in the immunoprecipitation. PCR products were separated by gel electrophoresis (6% PAGE), and visualized by PhosphorImager scanning of dried gels., ChIP-Seq libraries were constructed using NEBNext™ Ultra DNA Library Prep Kit for Illumina™ (NEB). Libraries were size selected for 200 bp DNA insert using AMPure™ XP beads (Beckman Coulter), PCR amplified and sequenced on HiSeq™ platform (Illumina). Raw reads were filtered and adapters were removed by FASTQC v0.10.1 using default parameters. The reads were mapped to the reference genome using bowtie v2 2.1.0. The non-unique reads were randomly distributed. Binding enrichment was called from the aligned reads using MACS2 2.0.10 and SPP ( using default parameters. All statistical analyses were performed using R., The 5΄ end hypermethylated spliced leader (SL) RNA Cap structure in T. brucei [m7G(5΄)ppp(5΄)m26 ApmApmCmpm3Ump] was detected using primer extension of a [γ-32P] 5΄-labeled oligonucleotide 5΄-CTGGGAGCTTCTCATACCCAATA-3΄ after hybridization to 2 μg total RNA from transgenic parasites before and after 2 days of tetracycline induction. Extension products, which are a mix of hypermethylated Cap1-4 species, were resolved by electrophoresis on a 10% polyacrylamide-7 M urea gel. Unmodified SL RNA (Cap0) was obtained from transgenic parasites treated w […]

Pipeline specifications

Software tools FastQC, Bowtie, MACS
Diseases Sleep Wake Disorders, Neurologic Manifestations, Parasomnias