To demonstrate general utility, we performed ChIP-seq according to this protocol for well-characterized factors such as TBP, H3K4me3, and TCF7L2/TCF4, one of the members of the Tcf/Lef family of Wnt pathway effectors [7], [8], [9].
We carried out DNase-seq (according to ENCODE standard protocols; see Methods and [ 22, 23] for mature mouse whole brain, and dissected cerebral cortex, cerebellum, and neural retina (age: 8-week adult), as well as specific ages of developing brain and retina.
We sorted the putative Myc target genes detected by ChIP-seq according to the strength of Myc binding (binding affinity) to their promoters and found, as expected, that the cluster containing genes that were transcriptionally induced upon Myc activation (up cluster #1) was significantly enriched for high-affinity Myc target genes (Table 2).
8 μg of total RNA was used as input material for the preparation of the RNA-Seq libraries, according to Illumina RNA Seq library kit with minor modifications.
Ruhleben's "Cineomatograph Theatre" [sic] was inaugurated on August 21st with a screening of a film by and about Ruhleben internees, featuring many "Camp celabraties" [sic] (Masterman Coll., Box 2 Seq. 422), according to the Captains' announcement.
The removal of the ribosomal component from the total RNA was performed using the RiboMinus Eukaryote Kit for RNA Seq (Invitrogen), according to the manufacturer's instructions.
Fragmented RNA was used to generate strand-specific RNA-Seq libraries according to the Directional mRNA-Seq Library Preparation Protocol (Illumina).
Poly(A)+ fraction was separated from 80 μg of total RNA using Oligotex mRNA Mini Kit (Qiagen, Valencia, CA) and fragmented using Fragmentation Reagents (Applied Biosystems/Ambion, Austin, TX) at 70° for 15 min. Fifty nanograms of the fragmented RNA was used to generate strand-specific RNA-Seq library according to the Directional mRNA-Seq Library Preparation Protocol (Illumina).
The RNA-seq libraries were constructed using a strand-specific RNA-Seq method according to Zhong et al. (2011).
For sequencing, 20 ng of the immunoprecipitated DNA was used to generate the ChIP-seq library according to the manufacture's protocol (Illumina) and sequenced with the Genome Analyzer II (Illumina).
(A ) PCA plot showing successful separation of RNA-seq data according to tissue but, importantly, not according to study.
