Harvesting mouse suprachiasmatic nucleus by vibrating microtome for diurnal transcriptome analysis

Summary The mammalian suprachiasmatic nucleus (SCN) is the principal circadian clock that synchronizes daily behavioral and physiological responses in response to environmental cues. Here, we present a protocol for harvesting mouse SCN by vibrating microtome for diurnal transcriptome analysis. We describe steps for mouse entrainment, isolation of the SCN, tissue preparation, slicing with a vibratome, and handling of the harvested SCN for RNA extraction. This protocol can also be used for harvesting other mammalian brain regions for genomic studies.


Highlights
Extraction of highly viable SCN tissue from the adult mouse brain using vibratome Precisely timed tissue harvest to detect circadian transcriptional rhythms Full description of mouse entrainment, SCN collection, and rhythmicity analysis A reference protocol for harvesting mammalian brain regions for genomic studies Institutional permissions (if applicable) All animal studies were performed under the guidance issued by Medical Research Council in Responsibility in the Use of Animals for Medical Research (July 1993) and Home Office Project Licence 19/0004.C57BL/6J were maintained and provided in-house by MRC Harwell.

Timing: 2 weeks
The animals are entrained under 12:12 h (hr) light-dark conditions.The number of mice to be used in the study should be carefully planned.For daily transcriptome profiling, we recommend harvesting mouse tissue(s) in 4-h interval over 24-h period.We used 3-4 biological replicates per time-point, where each biological replicate was a pool of four individual tissues (SCN).In total, we entrained and used ca.96 animals, composed of equal number of males and females for this study.
1. Singly house adult mice (8-12 weeks old) in individually ventilated cages fitted with running wheels, under 12:12 h (hr) light-dark (LD) conditions ($150 lux) with food and water available ad libitum. 1 Alternatively, if space and time are limiting, mice strains (such as C57BL6/J) that are known to entrain efficiently to an exposed LD cycle can be group housed in conventional cages inside a light-controlled circadian chamber (LCC).a. Place 8-10 cages in a specialized light-controlled circadian chamber 2 (air flow, temperature and humidity maintained according to institutional recommendations) programmed for lights on at 7 am (Zeitgeber time; ZT = 0) and lights off at 7 pm (ZT = 12) as shown in Figure 1.
b. Use a staggered light schedule when using multiple light-controlled circadian chambers for the ease of harvesting tissue(s) at distinct times during the day.
Note: For collecting tissues at night times, animals can be placed in cages inside a LCC programmed for lights on at 6 pm (ZT0) and lights off at 6am (ZT12).In this instance, for example, tissue(s) can be harvested from animals entrained at 8 am corresponding to ZT14.
2. Entrain mice to the 12:12 h light-dark schedule for 8-10 days.Change cage bedding on day seven if required.
CRITICAL: While the animals are under a light-dark schedule, minimize noise and cage disturbance as much as possible while maintaining animal welfare.It should be noted that light during lights-off would have a particularly detrimental effect on entrainment/experiment.

Circadian behavior analysis (optional)
Timing: 1 h 3. Analyze the locomotor activity (wheel-running activity) of the entrained mice for last 7-10 days (Figure 2).However, this step is not essential when working with a typical mice strain such as C57BL6/J, that can easily entrain to a LD cycle within one week.a. Use ClockLab Analysis 6 software (https://actimetrics.com/products/clocklab/) 3 to inspect the behavioral rhythm as per manufacturer's guidelines at 24 h period.
Note: While the data and protocol outlined here utilizes ClockLab software to analyze wheel running activity, any in-cage activity monitoring system which assesses activity in relation to the timing of the light cycle can be used.Mice are nocturnal animals and show maximum activity in the dark phase; ZT12-ZT24.
4. Discard animals that do not show robust rhythmic activity from the study.
CRITICAL: Use consistent analysis settings such as percentile/normalized actogram while assessing the circadian behavior using ClockLab.Note: The solutions are stable at 22 C-25 C, and there are no particular time constraints regarding their preparation or use.

KEY RESOURCES
CRITICAL: Diethyl pyrocarbonate (DEPC) is harmful with acute toxicity, Oral (Category 4), H302.Work under the fume hood and wash skin thoroughly after handling.Do not eat, drink or smoke when using this product.

STEP-BY-STEP METHOD DETAILS Brain dissection
Timing: 2 days The brains are harvested from the entrained mice at designated time-points after cervical dislocation.Approximately 16 mice comprising of four biological replicates (3-4 animals per biological replicate) are used per time-point.Note: It is important to collect the tissue at a particular time-of-the-day even if it is not used for circadian studies as the transcript abundance greatly varies with time.
4. Place brain, cortex down, upon the foil square on dry ice.a.If regions of the brain other than the SCN are to be used in subsequent analysis, the orientation of the brain on the foil may need to be changed so that the region of interest is not in direct contact with the foil.See 'troubleshooting' section for details. 5. Allow the brain to freeze for 5 min.During this time, further dissections can be performed.(Fig- ure 3).6.Once frozen, store the brain at À80 C until ready to continue to SCN dissection as specified below.
CRITICAL: For dark-phase time points, perform the brain dissection under dim red light (no more than 50 lux).Desired intensity of the red light is maintained using filters blocking wavelengths below 600 nm.Avoid any exposure of light whilst collecting and freezing the brains.

Timing: 2 weeks
The dissected brains are placed on dry ice and sliced in the coronal plane into 250 mM thick sections using a vibrating microtome.The brain slice between Bregma À0.3 mm and À0.8 mm, 6 guided by the anterior anchor points with the intact optic chiasm, third ventricle and SCN is selected for dissection under chilled conditions.
7. Carry the stored mouse brains from À80 C freezer to the desired dissection area using a dry ice tray.a. Place brain matrix, sterile razor blades, forceps, dissection needles etc. on dry ice to chill.8. Wipe the bench area, vibratome, light-dissection microscope etc. with RNaseZap to avoid contamination or degradation from RNAses.
CRITICAL: Wear gloves throughout the procedure and periodically change or wipe with RNAseZap.
9. Prepare the vibratome (https://campdeninstruments.com/products/7000smz-2-vibrotome) for coronal sectioning by following the specified guidelines in the equipment set up.Switch on the machine with the light source and scroll to the desired user and press [MENU].The sectioning parameters associated with the user will display on the screen.a. Add ice to the tissue bath cooling jacket of the vibratome.b.Fill the metal tray with chilled 1% RNAse free PBS solution.See 'troubleshooting' section for details.c.Cover the vibratome metal chuck (removable tissue support) with tape.Use super glue to stick 1 3 1 cm block of 3% agarose gel at the curved edge of the chuck.This provides support for the mouse brain during gradual sectioning.10.Separately, place the mouse brain (rostral to caudal) on the brain matrix and cut approximately 3 mm from the caudal end by razor blade to remove the cerebellum.
Note: Ensure the tissue is perpendicular to the brain matrix and the cutting line is in parallel with the grooves in the brain matrix.In doing so, it keeps the left and right hemispheres aligned on the same coronal section in order to minimalize angular variation in the dissection plane.This is especially important for non-circular and small nuclei.
11. Apply a tiny drop of superglue on the metal chuck.Now, gently transfer the brain from the matrix to the metal chuck with the rostral end facing upwards and affix the chuck on the metal tray as specified in the instruction manual https://lafayetteinstrument.com/downloads/manuals/7000-v3.0%20Eng.pdf.

CRITICAL:
The metal chuck should be submerged in the RNAse free PBS, covering the top of the brain specimen.
12. Manually set the bath to the start height following the manufacturer instructions.13.Bring the cutting blade to a suitable position by setting the start position using [ADVANCE] and the rotary knob.14.Press [SLICE ON/OFF] to begin and stop the sectioning.
a.After a slice is cut, press [RETURN] to slide the blade back to the start position (Figure 5A).b.Section the mouse brain from rostral to caudal up to approximately 1.2 mm of Bregma.
Note: Press [SLICE ON/OFF] to stop the sectioning once the blade reaches the top of the specimen, near the agarose block.After pressing [RETURN], if the slice is still attached to the specimen, dislodge it in the buffer gently with the help of a paint brush.
15. Gently transfer the mouse brain slice(s) with the help of a paintbrush onto the glass slide placed over a cold block.16.Examine the mouse brain slice(s) collected between Bregma À0.3 mm and À0.8 mm for the presence of intact bilateral SCN (Figure 4) using the cell density contrast under light microscope.See 'troubleshooting' for more details.17.Select the appropriate brain slice and tease apart the SCN from the surrounding hypothalamic tissue using pre-chilled dissection needles and forceps (Figure 5).18. Aliquot 10 mL of Buffer RLT (RNeasy Micro Kit, supplemented with b-mercaptoethanol) on to the sub-dissected SCN and transfer into 1 mL RNAse free microfuge tubes (kept on ice) using 10 mL pipette tips.19.Place the tube on dry ice.

Pause point:
The dissected SCN can be stored in Buffer RLT for long term at À80 C.

Timing: 3 days
The harvested SCN tissues are used for total RNA extraction to conduct polyA library preparation and sequencing.To increase the yield, SCN tissues collected at a single time-point are pooled prior to performing RNA extraction and purification.Post quality assessment, the extracted RNA is subjected to polyA library preparation and sequencing on Illumina platform.CRITICAL: The tissue samples should be thawed completely on ice before transferring to another tube.Special care should be taken while pooling the samples to avoid crosscontamination.
21. Adjust the sample volume to 350 ml with Buffer RLT and follow steps for RNA extraction using an RNeasy micro kit (Qiagen) as per the manufacturer's instructions (https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/ rneasy-kits) for purification of total RNA from microdissected cryosections using laser microdissection systems.The extracted RNA is finally eluted in Pause point: The extracted RNA can be stored for long term at À80 C.
22. Inspect the quality and quantity of the extracted RNA for each sample on Bioanalyzer using Agilent RNA 6000 pico chip.For library preparation, do not include any RNA sample with RIN (RNA integrity number) < 7.0 (Figure 6).See 'troubleshooting' for more details.
Note: Alternatively, RNA gel electrophoresis can be used to check for the integrity of the RNA, and Nanodrop to assess the purity and the concentration of the extracted RNA.Ideally, 260/ 280 ratio of $2.0 from Nanodrop is accepted as ''pure'' for RNA.

EXPECTED OUTCOMES
The anatomically deep-seated SCN can be successfully harvested from the mouse brain using correct anchor points, optimal section settings and temperature control as described in this protocol.Slicing the frozen mouse brain tissue using ultra slow vibration speed (0.07 mm/s) eliminates the risk of accumulating uneven or broken sections and offers great advantage over cryostat sectioning when operated manually or at pre-programmed settings.In principle, this method of tissue dissection can easily be adopted to isolate any brain region of interest using cell density contrast.Further processing of the sub-dissected SCN tissues in RNAse free conditions will yield approximately 100 ng high quality RNA (RIN > 8.0) at a concentration of > 10 ng/ul.The extracted RNA from each sample when subjected to library preparation and sequencing would potentially result in > 40 million reads which is optimal to conduct diurnal transcriptome analysis to investigate cycling gene expression in the SCN as represented in Figure 7.

QUANTIFICATION AND STATISTICAL ANALYSIS
Timing: 2 days Here we provide steps to process the RNA-Seq data using Galaxy 9 (https://usegalaxy.eu/)and identify transcripts with daily rhythmicity in the SCN using the R package dryR (Differential RhythmicitY analysis in R). 5 To execute dryR, the user will be required to install R for the appropriate operating system at https://www.r-project.org/.2. Align the quality checked and trimmed reads to mm10 genome assembly using STAR  3. The gene counts generated in the above step are used to assess rhythmicity by R package dryR (Differential RhythmicitY analysis in R). 5 dryR framework can analyze rhythmicity for one or more conditions.It can detect rhythmicity in a single condition (using the plot_single_cond() function) and compare rhythmicity across two or more conditions (using the dryseq() function).Alternatively, limma-voom method 12 from the Bioconductor package-limma (v3.48.0) can be used to quantify differential gene expression, and the resulting normalized logarithmic CPM values can serve as an input for rhythmicity detection by applications such as ECHO, 13 JTK_CYCLE 14 etc. 4. Prepare the data matrix file from the raw count data (e.g., Table 1) to serve as an input file for dryR package available at https://github.com/naef-lab/dryR .In this instance, gene counts from four biological replicates per time-point (ZT2, 6, 10, 14, 18, 22) are used.Execute dryR specifying the condition(s) and time-series as instructed in the package guidelines.See 'troubleshooting' for more details. 5.The output file will be .csvfile specifying the corresponding amplitude (amp), phase etc. for each transcript (e.g., Table 2).

LIMITATIONS
Here we describe how to collect SCN during the day at distinct time-points, with a large number of biological replicates per time-point.Until now, the preferred method for collecting tissues like the SCN is either by tissue punch 15 or laser capture microdissection (LCM). 16While the tissue-punch method often carries the risk of contamination from nearby tissue(s) and lack the desired precision, LCM is expensive and laborious especially when adopted for collection from large cohorts to achieve reasonable biological/technical replicates.For that reason, our protocol strikes a desired balance of collecting SCN tissues using low cost vibratome and dissection microscopes.Although this method is better over than the conventional tissue punch strategy, it still carries a minimal risk of contamination from nearby hypothalamic tissue.
In order to mitigate this risk, staining might help to locate specific brain regions, but the additional use of reagents and handling time has proven to be detrimental for high quality RNA extraction.Thus, this method of identifying SCN by cell density contrast between target and surrounding regions offer a huge benefit when performed cautiously.To maintain the integrity of the tissue, both sectioning and sub-dissection of the SCN from the mouse brain slice has to be performed side-by-side, not allowing for any pauses.This might result in lengthy processing time, but will not compromise the quality (and quantity) of the extracted genomic material.Thus, harvesting SCN for downstream RNA extraction for a large number of biological replicates, covering distinct times of the day, may require few weeks.

Figure 1 .
Figure 1.Mice in individual ventilated cages in light-controlled circadian chambers (A) Outline of circadian chambers.Mouse cages are placed inside light tight chambers with a light source above them.(B) Image of circadian chamber in use.(C) Detail of mouse cages inside circadian chamber.

Figure 3 .
Figure 3. Dissected mouse brain on dry ice 20. Thaw the SCN tissues containing 1.5 mL tubes on ice for RNA extraction.Pool 3 individual SCN tissue samples suspended in Buffer RLT, collected at the same ZT in one separate RNAse free tube.This is referred as one biological replicate (BR) and labeled (for example; ZT2_BR1).

Figure 5 .
Figure 5. SCN sub-dissection (A) Mouse brain sectioning using vibratome under chilled conditions.(B) Microscope view of 250 mM mouse brain slice with the intact SCN.(C and D) Marked boundary of the SCN region (blue) dissected under microscope.

Figure 6 .
Figure 6.RNA quality assessment using Bioanalyzer Example of bioanalyzer report for quality assessment of the extracted RNA samples (lane numbered 1-11) along with the ladder (left, L).The two bands denote 28S (top) and 18S (bottom) rRNA.

Figure 7 .
Figure 7. Rhythmic gene expression in the SCN (A-C) Representative examples of genes expressed in the SCN with in-situ hybridization images from Allen Brain Atlas (image credit: Allen brain atlas, Allen Institute 8 ) (left panel) and gene transcription levels (logarithmic normalized counts) over 24 h (right panel) for (A) Nr1d1, (B) Creb3l1, (C) Per2.Data are represented as G SEM.

TABLE Figure 2
. Circadian behavior analysis Double-plotted actogram showing wheel running activity for 2 weeks (y axis = number of days) in a 12:12 h light (yellow) /dark (white) cycle.Each horizontal line represents 48 h of activity and activity is represented by vertical bars.
14 ml elution buffer supplied with the kit.During the RNA extraction, perform on-column DNase digestion as per manufacturer's guidelines.

Table 1 .
Example of count data as input to dryR

Table 2 .
Example of output file for one condition using the dryR function plot_single_cond?This study did not generate new unique reagents.