RNA Detection

2 Materials

2.1 In Situ
Hybridization (ISH)

For ISH, researchers are free to use their favorite established

protocol.

2.2 Imaging of
the ISH Samples

1. Since large numbers of samples need to be imaged for the
   construction of the ISH atlas, access to a microscope with
   automatization possibility is advisable. We use Leica SP8 with
   40
   oil immersion objective, and use the mark-and-find func-
   tion for automated serial acquisition of image stacks.

2.3 Generation of a
Gene Expression Atlas

1. Fiji [1].


2. Python.


3. ITK [2].


4. R-Bioconductor [3] software.


5. Obtain the scripts published in Asadulina et al. 2012 [4] and
   Vergara et al. 2016 [5].

2.4 Single-Cell
mRNA-Sequencing
(scRNAseq) and
Mapping the
scRNAseq Data
to the Reference
Transcriptome

For the scRNAseq, the researcher is free to use any available

approach (seeNote 1) Here, we have used the C1 Single-cell

Auto Prep platform by Fluidigm.

1. Obtain the C1 Single-cell Auto Prep IFC for mRNA-seq,
   designed to fit your cell size range (seeNotes 2and 3 ).


2. Obtain the following kits and reagents: SMARTer Ultra Low
   Input RNA Kit and ADVANTAGE-2 PCR kit (Clontech),
   ERCC spike-in RNA (Ambion,) Nextera XT DNA Sample
   Preparation kit (Illumina)


3. Installbowtie2[6] andHTseq1[7].

2.5 Spatial Mapping
Tools

1. InstallR[3].


2. TheRscripts for spatial mapping are published in Achim et al.
   (2015) [8].
   Download these scripts via the following link:https://github.
   com/jbogp/nbt_spatial_backmapping.


3. TheBioWeb3D[9] based interface for the visualization of the
   reference voxels and spatial mapping results can be accessed via
   the following link: http://www.ebi.ac.uk/~jbpettit/map_
   viewer/

2.6 Data
Management Solutions

1. Since significant amounts of raw metadata will be produced and
   processed, a strategy for the storage, management, and stream-
   lined data processing should be developed, preferably prior to
   starting with the experiments.

112 Kaia Achim et al.