BLUEPRINT will be releasing all standardized protocols which will be used for the project as soon as they become available. Protocols will be updated when changes are introduced.
Protocols for sample collection and preparation
The protocols below have been used for all BLUEPRINT samples generated so far.Monocyte isolation
Cord blood progenitor sorting
CD4 naive cells
ChIP Cell fixation
Moreover it is important to note that the cells used so far, need a more stringent sonication method (see chromatin preparation protocol). To be more stringent, a lysisbuffer with 1% SDS instead of 0,15% SDS is used.
Protocols for ChIP analysis
It is important to note that 2 different ChIP methods have been used so far. The main differences between the two methods are the incubation time with the beads and the wash steps after chromatin/antibody/beads interaction. In the Histone ChIP protocol the incubation time with the beads is only 1 hour in comparison to overnight incubation in the Transcription Factor protocol. Increased incubation time with the beads in the Transcription Factor protocol will result in more chromatin/antibody/beads complexes and thus requires a more stringent wash procedure. This more stringent wash procedure is the other difference to the Histone protocol.
Sine July 2014, a new Histone ChIP protocol is being used. One ChIP contains 500.000 cells with a small amount of antibody. This decreases the background signal, while positive signal increases. The yield after such a ChIP will not always be measurable with Qubit, but with the new library preparation method from KAPA (KAPA Hyper Prep Kit) it is possible to generate a successful library for sequencing.
When using the old ChIP protocol, different ChIP methods were used for different cell types (see below). When using the new protocol, all cell types are treated in the same way.
For the different cell types, different ChIP methods are used as described below:
– For K562 cells all the histone marks are processed in the Transcription factor protocol.
– For Neutrophils all histone marks are processed in the Histone protocol.
– For Monocytes the H3K4me3 and H3K27ac are processed in the Histone protocol and the other marks (H3K36me3, H3K4me1, H3K27me3 and H3K9me3) in the Transcription factor protocol.