Protocols

ImmGen labs follow a rigorous process for data generation to maximize comparability between datasets of different origins. These protocols allow the derivation of reliable datasets from low numbers of highly purified cells directly ex vivo.

Most are generated from 5 week-old C57Bl/6J mice, shipped from the Jackson Laboratory one week prior to use (minimizing local environmental effects). In some specific cases, transgenic or KO mice are used as an alternative. Other inbred mice profiled also originate from the Jackson Laboratory (in collaboration with the Mouse Phenome Database project). Some use knockout mouse lines maintained in ImmGen labs (with matched littermates as controls).

All samples are sorted in ImmGen labs with a common Cell Preparation and Sorting protocol, with 2 successive sorts to maximize purity (most often 2 flow cytometry sorts), the final sort being directly into cell lysis on fixation buffer, depending on the protocol. These are then shipped to the ImmGen core team for processing using single microarray, RNAseq or ATACseq pipelines.

ImmGen Protocols:

- Cell Preparation and Sorting

- Total RNA extraction (Microarray)

- Poly-A+ RNA Sequencing (TruSeq)

- Ultra-Low-Input (ULI) RNA-seq

- Standard ImmGen 14-cell Set

Gencode

ImmGen currently uses GENECODE annotations version M18 for single cell analysis and M16 for all other data.