Cerebral Ventricles

The Iso-Seq method for sequencing full-length transcripts was developed by PacBio during the same time period as the genome assembly. We therefore used this technique to improve characterization of transcript isoforms expressed in cattle tissues using a diverse set of tissues collected from L1 Dominette 0 1449 upon euthanasia. The data were collected using an early version of the Iso-Seq library protocol [26 ] as suggested by PacBio. Briefly, RNA was extracted from each tissue using Trizol reagent as directed (Thermo Fisher). Then 2 μg of RNA were selected for PolyA tails and converted into complementary DNA (cDNA) using the SMARTer PCR cDNA Synthesis Kit (Clontech). The cDNA was amplified in bulk with 12–14 rounds of PCR in 8 separate reactions, then pooled and size-selected into 1–2, 2–3, and 3–6 kb fractions using the BluePippin instrument (Sage Science). Each size fraction was separately re-amplified in 8 additional reactions of 11 PCR cycles. The products for each size fraction amplification were pooled and purified using AMPure PB beads (Pacific Biosciences) as directed, and converted to SMRTbell libraries using the Template Prep Kit v1.0 (PacBio) as directed. Iso-Seq was conducted for 22 tissues including abomasum, aorta, atrium, cerebral cortex, duodenum, hypothalamus, jejunum, liver, longissimus dorsi muscle, lung, lymph node, mammary gland, medulla oblongata, omasum, reticulum, rumen, subcutaneous fat, temporal cortex, thalamus, uterine myometrium, and ventricle from the reference cow, as well as the testis of her sire. The size fractions were sequenced in either 4 (for the smaller 2 fractions) or 5 (for the largest fraction) SMRTcells on the RS II instrument. Isoforms were identified using the Cupcake ToFU pipeline [27 ] without using a reference genome.
Short-read–based RNA-seq data derived from tissues of Dominette were available in the GenBank database because her tissues have been a freely distributed resource for the research community. To complement and extend these data and to ensure that the tissues used for Iso-Seq were also represented by RNA-seq data for quantitative analysis and confirmation of isoforms observed in Iso-Seq, we generated additional data, avoiding overlap with existing public data. Specifically, the TruSeq stranded mRNA LT kit (Illumina, Inc.) was used as directed to create RNA-seq libraries, which were sequenced to ≥30 million reads for each tissue sample. The Dominette tissues that were sequenced in this study include abomasum, anterior pituitary, aorta, atrium, bone marrow, cerebellum, duodenum, frontal cortex, hypothalamus, KPH fat (internal organ fat taken from the covering on the kidney capsule), lung, lymph node, mammary gland (lactating), medulla oblongata, nasal mucosa, omasum, reticulum, rumen, subcutaneous fat, temporal cortex, thalamus, uterine myometrium, and ventricle. RNA-seq libraries were also sequenced from the testis of her sire. All public datasets, and the newly sequenced RNA-seq and Iso-Seq datasets, were used to annotate the assembly, to improve the representation of low-abundance and tissue-specific transcripts, and to properly annotate potential tissue-specific isoforms of each gene.

TAZ-KO mice were described previously (55 (link)). Adult (9–15 months of age) male TAZ-KO mice and their male wildtype littermates were sacrificed by CO₂ narcosis and cervical dislocation. Heart tissues (ventricle) were dissected, rinsed in PBS, snap frozen in liquid nitrogen, and stored in a −80 °C freezer for later use. To isolate total protein and RNA, 30 to 40 mg of tissue was homogenized by sonication in RIPA buffer on ice. After centrifugation, the supernatants containing protein were used for immunoblotting analysis, whereas tissue pellets were used to extract total RNA using the RNeasy Plus Mini Kit (QIAGEN).

Apple and Samsung devices were used for image acquisition. Unless otherwise noted, all image acquisition was performed using an iPhone XR (Apple). For convenience, time-lapse videos of the embryonic zebrafish heart were acquired using the ProCam 8 app, which allowed the 1X lens to be locked for use, and digital zoom extended to 4.0X. Video acquisition at 1080p resolution and 60 frames per second (fps) was used because 4 k resolution or higher frame rates decreased fluorescence sensitivity and signal:noise ratio. All images and videos were transferred to a laptop using Airdrop (Apple) or a USB cable to avoid video data compression. Videos were imported into Fiji^{14 (link)} by first converting videos to a series of TIFF images (one per frame) using Adobe Photoshop. After opening smartphone videos in Photoshop, images were cropped and then saved as an image sequence using the File Export Render Video Photoshop Image Sequence (TIFF format) command. The image sequence was then opened in Fiji through the File Import Image Sequence command. For non-fluorescent time-lapse viewing of paramecia, zebrafish swimming, and tadpole and caterpillar escape responses, we used 1080p resolution with 120 fps acquisition.
For zebrafish heart videos, detection and measurement of heart chamber movements in some videos was aided by edge detection. In these instances, Fiji was used to detect edges using the Process Find Edges command. To detect fluorescence changes associated with atrium and ventricle movements, the raw or edge-detected image sequence was used and further analyzed within Fiji. First, the Analyze Set Measurements command was used to instruct Fiji to measure minimum and maximum gray values, mean gray values, and limit to threshold. The image stack was converted to greyscale using the Image Type 8-bit command. In instances where image drift or non-biological movements occurred, the Plugins Image Stabilizer command was used in attempt to eliminate this issue. The image was scanned for potential regions of interest (ROI) where the heart chamber walls consistently moved back and forth across the x–y axis. Once a ROI was defined, it was added using the Analyze Tools ROI manager window or the “t” shortcut. Concurrently to identifying a ROI, the Image Adjust Threshold window was used to establish a threshold whereby the moving chamber walls consistently moved into and out of the ROI box. Once an ideal combination of image threshold and ROI box position was established, the image was converted to binary data using the Process Binary command selecting default, dark, and black background settings. To measure fluorescence intensity within the ROI over time, Multi Measure tool within the ROI manager (under ‘More’ tab) was used to create a set of measurements over time. Raw data were cut and pasted directly into a Microsoft Excel spreadsheet. Within the spreadsheet, formulas were used to identify the onset of each heartbeat using the maximum intensity column, and plots were made using GraphPad Prism 7.0. The onset of each beat was defined by the transition from 0 to 255.