Zhuang Research Lab

MERFISH Data and Protocols

MERFISH is an imaging method capable of simultaneously measuring the copy number and spatial distribution of hundreds to thousands of RNA species in single cells. This technique was introduced in the paper:

Spatially resolved, highly multiplexed RNA profiling in single cells
K.H. Chen, A.N. Boettiger, J.R. Moffitt, S. Wang, X. Zhuang

Science

348

aaa6090 (2015)

We have introduced a revised set of MERFISH protocols that allow the number of measured cells to be increased substantially. These methods are described here:

High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization
J.R. Moffitt, J. Hao, G. Wang, K.H. Chen, H.P. Babcock, X. Zhuang

PNAS

113

11046 (2016)

We recently introduced a sample clearing approach customized for smFISH and MERFISH. This approach and the performance enhancements it provides are described here:

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing
J.R. Moffitt, J. Hao, D. Bambah-Mukkua, T. Lu, C. Dulac, X. Zhuang

PNAS

113

14456 (2016)

Additional Data and Resources

MERFISH methods
A Methods in Enzymology paper describing in greater detail the protocols necessary to perform a MERFISH experiment.

Example MERFISH Data 1
A set of example raw MERFISH data, analyzed data, and various files useful in the analysis of MERFISH data. The barcode sets that were used for the 14-bit MHD2 and 16-bit MHD4 codes are also included.

Example MERFISH Data 2
A set of files used to demonstrate the use of the fast and efficient probe design pipeline introduced in:

High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization
J.R. Moffitt, J. Hao, G. Wang, K.H. Chen, H.P. Babcock, X. Zhuang

PNAS

113

11046 (2016)

MERFISH Analysis Software
All of the MERFISH software is now available on github, including example scripts that utilize the example data above.

IMR90 RNA-Seq
The RNA-seq data to which we compared MERFISH.

All identified RNA molecules in the 140-gene measurements
This excel table contains properties of all identified RNA molecules in the 140-gene experiments using the 16-bit MHD4 code. Experiment is a unique index assigned to independent experiments on different days. Codebook specifies the version of the two shuffled 16-bit MHD4 codebook used. Cell_ID represents a unique index assigned to each imaged cell in each experiment. intCodeword is an integer representation of the binary barcode observed for that RNA. Gene_Name is the name of the assigned gene. Exact_Match is 1 only if the word is an exact match to an RNA code word. Corrected_Match is 1 only if the word is a corrected match with one-bit error originally. Cell_Position_X and Cell_Position_Y represent the position of the cell within the sample measured in microns. RNA_Position_X and RNA_Position_Y represent the centroid position of the each RNA spot measured in nanometers.

Example MERFISH Data from High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization
This zip file contains measured RNAs, cell boundaries, and the codebook associated with these measurements.

Example MERFISH screening data from High-throughput, image-based screening of pooled genetic variant libraries
This zip file contains raw image data for all imaging rounds for a subset of positions from the mMaple3+/mMaple3- experiment in addition to the barcode-to-genetic-variant lookup table, position list, analysis parameters, and the data for flat field normalization.