DOI: 10.5061/dryad.sj3tx96g3
title: Data from: Protocol optimization improves the performance of multiplexed RNA imaging

description: Spatial transcriptomics has emerged as a powerful tool to define the
 cellular structure of diverse tissues. One such method is multiplexed
 error robust fluorescence in situ hybridization (MERFISH). MERFISH
 identifies RNAs with error tolerant optical barcodes generated through
 sequential rounds of single-molecule fluorescence in situ hybridization
 (smFISH). MERFISH performance depends on a variety of protocol choices,
 yet their effect on performance has yet to be systematically examined.
 Here we explore a variety of properties to identify optimal choices for
 probe design, hybridization, buffer storage, and buffer composition. In
 each case, we introduce protocol modifications that can improve
 performance, and we show that, collectively, these modified protocols can
 improve MERFISH quality in both cell culture and tissue samples. As RNA
 FISH-based methods are used in many different contexts, we anticipate that
 the optimization experiments we present here may provide empirical design
 guidance for a broad range of methods.  The data provided in
 these repository are those used in the optimization and validation of
 these protocols. 
description: # Data from: Protocol optimization improves the performance of multiplexed
 RNA imaging ## File Organization The repository has two downloadable zip
 folders. * readout_screening.zip contains the Bit_Screening folder. *
 data.zip contains all other folders. ### U2OS_MERFISH This folder contains
 data associated with MERFISH measurements of 130 RNAs in U-2 OS cell
 culture with old and optimized protocols. It contains the following files:
 barcode_metadata_Rep5_Ctrl.csv -- The identified RNAs from one replicate
 measurement of U-2 OS with the old protocols barcode_metadata_Rep5_Opt.csv
 -- The identified RNAs from one replicate measurement of U-2 OS with the
 optimized protocols barcode_metadata_Rep6_Ctrl.csv -- The identified RNAs
 from one replicate measurement of U-2 OS with the old protocols
 barcode_metadata_Rep6_Opt.csv -- The identified RNAs from one replicate
 measurement of U-2 OS with the optimized protocols
 replicate_watershed_counts.csv -- The number of nuclei counted in each of
 the above experiments FPKMData.matb (.csv) -- The abundance associated
 with each RNA determined by bulk RNA sequencing in a closed format (matb)
 or an open format (csv) barcode_metadata files have the following fields *
 barcode_id: index of the associated barcode * gene_name: name of the
 identified gene * slice_id: an index associated with different ROIs in the
 sample. Always 1 for these samples. * fov_id: an index for the field of
 view in which the RNA was imaged * total_magnitude: the sum of all
 normalized brightness values for pixels located within a region identified
 as a molecule * brightness: the average brightness across all pixels
 assigned to an RNA * area: the number of pixels associated with the
 molecule * abs_position_1: the x position of the molecule centroid on the
 stage (µm) * abs_position_2: the y position of the molecule centroid on
 the stage (µm) * abs_position_3: the z position of the molecule centroid
 as determined by the nanopositioner elevation * error_bit: the bit number
 if an error has been identified in the barcode. If no bit number is
 applicable, this value is 0 * error_dir: the direction of the error
 applied. If 0-&gt;1: 0. If 1-&gt;0: 1 replicate_watershed_counts has the
 following fields: * sample: the name of the sample/replicate *
 nuclei_counts: the number of nuclei identified in each sample FPKMData has
 the following fields * geneName: the name of the gene * id: the id of the
 RNA isoform targeted * FPKM: the abundance determined by bulk RNA
 sequencing ### Colon_MERFISH This folder contains data associated with
 MERFISH measurements of ~1000 RNAs in Swiss rolls of the mouse colon with
 the old and optimized protocols. It contains the following files:
 barcode_metadata_old.csv -- The identified RNAs from a Swiss roll
 measurement with the old protocols barcode_metadata_optimized.csv -- The
 identified RNAs from a Swiss roll measurement with the optimized protocols
 barcode_metadata files have the same fields as described above ###
 U2OS_smFISH This folder contains data associated with all of the smFISH
 molecules identified in the optimization experiments presented in this
 work The following files describe the following experiments: *
 ‘MoleculesID_SingleFrame_AdenineSpacers’ examines the impact of
 hybridizing probes with differing lengths of spacer nucleotides between
 the encoding and readout regions. The sample names are given by the length
 of adenine spacers placed between the encoding and readout 1 regions, and
 between the readout 1 and readout 2 regions. *
 ‘MoleculesID_SingleFrame_Annealing_ComplexOpool’ examines the impact of
 annealing on hybridization using a complex 130-RNA species library. The
 sample names are given by the heat at which the sample was annealed, or
 No_Annealing for not undergoing annealing. *
 ‘MoleculesID_SingleFrame_Annealing_GeneOpool’ examines the impact of
 annealing on hybridization using small single-gene oligopools. The sample
 names are given by the heat at which the sample was annealed, or
 No_Annealing for not undergoing annealing. *
 ‘MoleculesID_SingleFrame_HybDur_ProbeConc’ examines the impact on
 hybridization of differing durations on different concentration of a
 complex 130-RNA species library. The sample names are given by the
 dilution from a 4 µM stock of the library concentration and the duration
 for which the sample was hybridized. For instance, '1-100_1-Day'
 was diluted 1:100 (ie: 40 nM) and hybridized for 1 day, while
 '1-1_7-Day' was the hybridized at the stock concentration (4 µM)
 for 7 days. * ‘MoleculesID_SingleFrame_ProbeLength_FormamideConc’ examines
 the impact on hybridization using probes of differing encoding regions
 lengths and also moderated by the concentration of formamide in the
 hybridization buffer. The sample names are given by their formamide
 concentration used in hybridization and encoding region length. For
 instance, '10%_20-nt' used 20-nt encoding regions hybridized at
 a 10% formamide concentration. *
 ‘MoleculesID_SingleFrame_ReadoutHybBufAging’ examines the impact of using
 a readout hybridization buffer and their respective readout probes that
 had been aged 1, 2, 4, or 7 days in advance under various conditions. The
 sample names are given by the duration that readout hybridization buffer
 was aged and the conditions in which this buffer was stored. For instance,
 '4-Day Mineral Oil' was stored for 4 days under a layer of
 mineral oil; '0-Day' was prepared fresh. These csv files contain
 the following columns: * x: the x position within the frame, given in
 pixels * y: the y position within the frame, given in pixels * value: the
 fluorescent intensity value of this molecule * sample: the identity of the
 sample conditions * frame_num: the number of the image frame in which this
 molecule was located * dye: the readout fluorophore dye indicating the
 laser channel fluorophore was read (AF488: 473 nm, ATTO565: 561 nm, Cy5:
 635 nm, AF750: 750 nm) * FOV_num: an index for the field of view in which
 this molecule was imaged * rep_num: the biological replicate identity for
 which this molecule was located * scope (optional): the identity of the
 microscope used to image this sample * gene (optional): if applicable, the
 name of the gene the oligopool was designed to hybridize to The following
 files describe the following experiments: *
 ‘MoleculesID__LongBleaching_AgedImgBuffer’ examines the impact of
 bleaching on imaging buffer that had been aged for 0 or 7 days. The sample
 names are given by the duration of imaging buffer aging. *
 ‘MoleculesID__LongBleaching_ImgBufpHOpt’ examines the impact of bleaching
 on fresh imaging buffer with different buffer compositions and pH when
 fully deoxygenated when continuously imaged for 250 seconds per dye per
 FOV. This examines the steady-state bleaching loss. The sample names are
 given by the buffer composition and pH that was modified. *
 ‘MoleculesID_ShortBleaching_ImgBufpHOpt’ examines the impace of bleaching
 on fresh imaging buffer with different buffer compositions and pH when
 imaging buffer is flowed and then imediately imaged over 81 FOVs with 2
 frames taken per FOV at 2fps. This examines the immediate fluorescent
 intensity loss during the oxygen-scavenging state. The sample names are
 given by the buffer composition and pH that was modified. *
 'MoleculesID_ShortBleaching_ATTO565_AF488' examines the impact
 of dynamic bleaching on ATTO565 and AF488 fluorophores. The sample names
 are given by the imaging buffer. *
 'MoleculesID_LongBleaching_ATTO565_AF488' examines the impact of
 static bleaching on ATTO565 and AF488 fluorophores. The sample names are
 given by the imaging buffer. These csv files contain the following
 columns: * x: the x position within the frame, given in pixels * y: the y
 position within the frame, given in pixels * sample: the identity of the
 sample conditions * dye: the readout fluorophore dye indicating the laser
 channel fluorophore was read (Cy5: 635 nm, AF750: 750 nm) * FOV_num: the
 sequential FOV for which this molecule was located * rep_num: the
 biological replicate identity for which this molecule was located *
 time#val: the fluorescent intensity value for this identified molecule, on
 the FOV_num listed, given by the sequential time point given by #. This is
 measured in seconds. The following files contain the following
 information: * 'WatershedCounts_Annealing_ComplexOpool' contains
 nuclei counts (i.e. cell numbers) for all listed experiments. *
 'WatershedCounts_Annealing_GeneOpool' contains nuclei counts
 (i.e. cell numbers) for all listed experiments. *
 'WatershedCounts_HybDur_ProbeConc' contains nuclei counts (i.e.
 cell numbers) for all listed experiments. *
 'WatershedCounts_ProbeLength_FormamideConc' contains nuclei
 counts (i.e. cell numbers) for all listed experiments. *
 ‘WatershedCounts_ReadoutHybBufAging’ contains nuclei counts (i.e. cell
 numbers) for all listed experiments. These csv contains the following
 fields * rep_number: a unique number for each biological replicate of an
 experiment * sample: a short descriptor of the experiment * nuclei_counts:
 the number of nuclei identified in each sample ### Scripts This folder
 contains example code associated with the identification of molecules and
 the quantification of their brightness in molecular counting and
 brightness measurements. It also contains code associated with the
 counting of nuclei from DAPI images. Note that this code is not intended
 to be run as is, as we do not provide example images, and the code assumes
 a variety of naming conventions and organization of the images, which we
 do not provide. However, we provide this code so that the image processing
 steps used to identify molecules are clear. These scripts are provided
 under the same CC0 license. They were used with python 3.6 and the
 following package versions: storm-analysis==2.1\
 tifffile=2019.7.26.2=py36_0\ pillow=7.2.0=py36hcc1f983_0\
 pandas=1.0.5=py36h47e9c7a_0\ numpy=1.17.0=py36h19fb1c0_0\
 matplotlib=3.2.2=0 ### Bit_screening This folder contains raw imaging data
 for bit screening experiments. Raw data is stored as tif files in the
 format generated by the storm control codebase
 ([https://github.com/MoffittLab/storm-control](https://github.com/MoffittLab/storm-control)) with associated metadata files. This folder contains the following sub folders * HEK * HeLa * Human_TrigeminalGanglia * Mouse_Colon * Mouse_Ileum * Mouse_Jejunum * Mouse_TrigeminalGanglia * U2-OS Each folder contains a rawdata folder(s) and a data_organization file(s) data_organization files describe the format of the raw data files as to be compatible with the Moffitt Lab storm analysis codebase ([https://github.com/MoffittLab/storm-analysis](https://github.com/MoffittLab/storm-analysis)). These files contain the following fields * bitName: readout probe name consistent with nomenclature of the corresponding manuscript * imageType: file naming structure for file base * imageRegExp: file naming structure combining file base, fov number, hybridization round, and camera id * bitNumber: readout probe number * imagingRound: hybridization round * color: excitation laser used (nm) * imagingCameraID: optional, if using a multi camera microscope, camera id * frame: frame number within tif stack * zPos: z position of objective as moved by nanopositioner * fiducialImageType: file naming structure for file base of fiducial channel used to warp succesive imaging rounds * fiducialRegExp: file naming structure combining file base, fov number, hybridization round, and camera id for fiducial channel * fiducialImagingRound: hybridization round of fiducial images * fiducialFrame: frame number within tif stack for fiducial chanel * fiducialColor: excitation laser used (nm) for fiducial channel * fiducialCameraID: optional, if using a multi camera microscope, camera id for fiducial channel