eamag/protocols-with-steps huggingface dataset:
TITLE: Immunofluorescence protocol of Pax7 and androgen receptor for frozen muscle sections with unmasking
AUTHORS: Hiroshi Sakai

[DESCRIPTION]
Immunofluorescence protocol of Pax7 on muscle tissues is a critical step for studying skeletal muscle regeneration. Here, I describe the simple protocol for Pax7 for isopentane-frozen muscle tissues with unmasking. Androgen receptor, a member of the nuclear receptor superfamily, can be stained with this protocol to see the double positive cells. 

[BEFORE_START]
REAGENT SETUP
Sodium citrate (10 mM, pH = 6)
            - A solution (Keep at room temperature)
                        - 2.1 g of Citric Acid Monohydrate (C6H8O7・H2O)
                        - 100 ml of H2O
            -B solution (Keep at room temperature)
                        - 14.7 g of Trisodium citrate dihydrate (C6H5Na3O7・2H2O)
                        - 500 ml of H2O
            - Sodium citrate
                        - 9 ml of A solution
                        - 41 ml of B solution
                        - 450 ml of H2O

[GUIDELINES]
Use freshly cut sections since Pax7 and AR on sections are not stable.

[STEPS]
SECTION: Fix
1. Fix the slides with 4% PFA/PBS for 5 min at room temperature.

 
SECTION: Fix
2. Wash the slides in PBS three times for 5 min.
SECTION: Unmasking
3. Place the slides into a slide chamber in PBS
SECTION: Unmasking
4. Fill a beaker with 500 ml of Sodium citrate.
SECTION: Unmasking
5. Place the beaker on the hotplate and turn it on full power.
 
SECTION: Unmasking
6. Once boiling, transfer the slides from PBS to the beaker.
 
SECTION: Unmasking
7. Boil the slides for 10 min.
SECTION: Unmasking
8. Remove the beaker from the hotplate and let the beaker cool for 20 min.
SECTION: Unmasking
9. Remove the slide chamber from the beaker.
SECTION: Unmasking
10. Wash slides in PBS three times for 5 min.
SECTION: Blocking
11. Block the slides with 5% Goat serum/0.05% Triton X-100/PBS for 1h at room temperature. Prepare 100 µl of 5% Goat serum/0.05% Triton X-100/PBS per slide.
SECTION: Blocking
12. Drain the slides for a few seconds (do not rinse) and wipe around the sections with tissue paper.
SECTION: Primary antibodies
13. Dilute primary antibodies as follows in 5% Goat serum/0.05% Triton X-100/PBS. Prepare 100 µl antibody solution per slide. Add primary antibody solution to the slides.
100 µL 
5 µL 
0.25 µL 

SECTION: Primary antibodies
14. Incubate overnight at 4°C.
SECTION: Primary antibodies
15. Wash the slides in PBS three times for 5 min.
SECTION: Secondary antibodies
16. Dilute secondary antibodies as follows in 5% Goat serum/0.05% Triton X-100/PBS. Prepare 100 µl antibody solution per slide. Add secondary antibody solution to the slides.
100 1
0.1 µL 
0.1 1
0.05 1

SECTION: Secondary antibodies
17. Incubate for 1h at room temperature protected from light.
SECTION: Secondary antibodies
18. Wash the slides in PBS three times for 5 min.
SECTION: Mount
19. Mount using ProLong™ Glass Antifade Mountant and add coverslips.
TITLE: Nebuloni, F. &amp; Do, Q. B. et al. (2024) A fluid-walled microfluidic platform for human neuron microcircuits and directed axotomy
AUTHORS: Federico Nebuloni, Quyen Do, Richard Wade-Martins

[DESCRIPTION]
This collection contains six protocols detailing methods used in Nebuloni, F. & Do, Q. B.et al. (2024) A fluid-walled microfluidic platform for human neuron microcircuits and directed axotomy.

[STEPS]
TITLE: Superior Nutra Keto 100% Safe And Effective FEATURES Of Ingredients
AUTHORS: SuperiorNutraKeto  

[DESCRIPTION]
Official Website - Click Here forSuperior Nutra Keto
Availability Of Superior Nutra Keto - Online On Website

Main Benefits - Fat & Weight Loss Without Any Side Effect

VISIT THE OFFICIAL WEBSITE OF POWER KETO GUMMIES TO ORDER

[STEPS]
TITLE: Qbiotix protocol
AUTHORS: vito 

[STEPS]
?. 1- collect saliva vial
1 mL
?. 2- open vial and scan barcode
?. 3- dispense   sample to single well in plate with a pipette
250 µl
?. 4- dispense   of magnetic bead solution in well with a pipette
250 µl
?. 5- incubate at controlled temperature on the plate reader
?. 6- place plate on magnetic separator
?. 7- insert plate on analyzer (reading speed depends on chosen plate reader instrument)
?. 6- read results
TITLE: Thawing of feeder-free hPSCs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the procedure of thawing feeder-free human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex 

General Notes
Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions.
We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two mediums are not interchangeable. Pick one and stick to it.
We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex--coated cell culture plates without observing obvious differences.


[STEPS]
1. Prepare one 6-well VTN/Matrigel/Geltrex-coated plate for each vial of frozen hPSCs. 

A detailed protocol on "Coating plates" can be found in the "Feeder-free culturing of hPSCs" collection. A link to this collection can be found in the title section of this protocol, located above

4. Centrifuge 200-300 x g, 5 min 

3. Pipette thawed cell suspension into 10 ml pre-warmed DMEM/F12.
2. Place vial of frozen hPSCs in 37 °C  water bath with constant agitation. 

8. Re-suspend the cells using a P1000 tip.
5. While cells are spinning, aspirate the VTN/Matrigel/Geltrex solution from the coated plates and add 2 ml pre-warmed Feeder-free medium + Rock inhibitor to each well. 
7. Add 1 ml Feeder-free medium + Rock inhibitor
6. Aspirate most of the medium on the centrifuged hPSCs, being careful not to disturb the pellet
11. Spread the cells by moving the plate in left-right, then backward-forward motion.
10. Check the cells under the microscope to get an idea of the resulting cell density.
9. Dispense the cells onto the VTN/Matrigel/Geltrex-coated plate, adding 160 µl per well.
13. Change 2 ml pre-warmed Feeder-free medium for each well every other day. When large colonies emerge or hPSCs density reaches 50-80%, passage using Accutase or ReLeSR. It usually takes 5-7 days for the thawed cells to grow to this point. 

A detailed protocol on passaging using Accutase or ReLeSR can be found in the "Passaging of hPSCs" protocol, in the "Feeder-free culturing of hPSCs" collection. A link to this collection can be found in the title section of this protocol, located above
12. Place the plate in the low oxygen incubator
5.1. Feeder-free Medium (version A)
     StemFlex   basal medium      450 ml        StemFlex   supplement      50 ml   
 
Feeder-free Medium (version B)
     mTeSR-plus   basal medium      400 ml        mTeSR-plus   supplement       100 ml   

 

Y-27632 (1,000X)
     Y-27632      5 mg        DMSO      1.56 ml   
 

 Feeder-free medium + Rock inhibitor
     Feeder-free   medium      50 ml        Y-27632   (1,000X)      50 ul
TITLE: AMAS  to Concatenate Sequences
AUTHORS: Dakota Betz

[DESCRIPTION]
Brief instructions on how to use the AMAS Package to concatenate sequences in the command line. Especially useful if your SequenceMatrix is no longer compatible with your version of Java.

[STEPS]
SECTION: Installation
1. Prerequisites: Python 3 previously installed.
Download the AMAS package here:
 
Once downloaded, drag the AMAS folder into your Applications folder. You will need the path to this folder in order to proceed.
SECTION: Concatenate
2. Use the command line to navigate to the folder containing your aligned files (one alignment per gene). 
 

SECTION: Concatenate
3. Use the concat command to concatenate your files: 
-i indicates the input files you want to concatenate
gene1.fas is your first aligned gene file (drag the file to the terminal to automatically include the path to it)
gene2.fas is your second aligned gene file (drag the file to the terminal to automatically include the path to it)
 
-f indicates the format of your files (in this case fasta, but you can also use nexus, phylip, etc)
-d indicates the data type (DNA in most cases, use aa for amino acids)
 
This will generate two files:
"concatenated.out" is your concatenated file. Add the file extension ".fas" so you can open it in AliView or Mesquite to check that it worked (recommend AliView if you're staying in FASTA format).
"partitions.txt" lists the partitions for your new concatenated file

 

SECTION: Additional Options
5. You can specify the format of the partitions file, depending on the program you want to use. For RAxML: 
 
If you open the partitions file, you can check that it is properly formatted. For RAxML, it should look something like this:
DNA, 16S = 1-605 
DNA, COX1 = 606-1200 
DNA, 28S = 1201-1800
SECTION: Additional Options
4. If you would like to change the output format (something other than FASTA) use the argument -u, as in:
TITLE: Anti-Neu5Gc Antibody Kit Protocol - Flow Cytometry
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Anti-Neu5Gc may be used for staining cells prior to analysis by Flow Cytometry. This kit contains all the essential components needed to identify Neu5Gc on the surface of cells by flow cytometry.</div><div class = "text-block">Use of the blocking agent (Neu5Gc Assay Blocking Solution) provided in the kit is essential, as commonly used blocking agents invariably contain serum, or serum components, that can either inhibit detection or introduce Neu5Gc contamination.</div><div class = "text-block">Tissue culture-grown CHO-K1 cells can be used as a positive control, and human peripheral blood mononuclear cells serve as a negative control. Adherent tissue culture-grown cells should be released from the culture flasks by using 5-10mM EDTA for 10 minutes at room temperature. Other non-enzymatic methods, such as Accutase®, may be used. Immediately wash cells in blocking buffer that contains a lower concentration of EDTA, and resuspend cells in blocking buffer to determine cell numbers and viability.</div><div class = "text-block">It is assumed that the user is familiar with the general principles and practices of Flow Cytometry.</div></div>

[STEPS]
?. 
?. Three (3) tubes containing cells to be examined. If choosing to use different dilutions of primary antibody, as noted in step 3, more tubes may be needed.Three (3) tubes containing positive control cells.Three (3) tubes containing negative control cells.
?. Each set of tubes to be analyzed should receive an antibody treatment as follows:Tube 1 will contain cells that will receive no antibody, to be used as unstained control (optional but highly recommended).Tube 2 will contain cells that receive Control Antibody.Tube 3 will contain cells that receive Primary Antibody.Note: The dilution of the antibody can range from 1:200 to 1:1,000. Investigator must determine optimum dilution for cells being analyzed.Tip: If this is the first time running this experiment, optimize by using different dilutions of primary antibody. More tubes containing sample will be needed.
?. Wash cells by adding 1ml cold PBS, then gently centrifuge at 4°C. Carefully remove supernatant and discard.
?. Gently resuspend cells in 100µl of the appropriate diluted antibody as outlined above.
?. Incubate cells on ice for at least 1 hour.
?. Wash cells as above.
?. Gently resuspend cells in 100µl Secondary Antibody in diluent buffer, and incubate for 1 hour on ice.Note: The dilution of the antibody can range from 1:200 to 1:1,000. Investigator must determine optimum dilution for cells being analyzed.
?. Wash cells as before.
?. Resuspend cell pellet in 400µl of diluent buffer.
?. Run cells through the flow cytometer.
TITLE: Protocol for Exo-CIP™ Rapid PCR Cleanup (#E1050)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Exo-CIP™ Rapid PCR Cleanup Kit
Rapidly degrade residual PCR primers and dephosphorylate excess dNTPs after amplification
Reaction complete in 4 minutes
Thermolabile formulation can be heat inactivated in 1 minute at 80°C
PCR product can be used directly in downstream applications
Compatible with commonly-used reaction buffers

[STEPS]
1. Transfer 5 µL to a new PCR tube and add 1 µL and 1 µL. The final volume is 7 µL.
2. Mix thoroughly and briefly centrifuge at 1000 x g.
3. Incubate the reaction tube for 4 min at 37 °C followed by 1 min at 80 °C.
4. Submit 3 µL or less* for sequencing using  BigDye™ Terminator v3.1 Cycle Sequencing Kit or store the treated samples at -20 °C  for longer term storage.
TITLE: Protocol for evaluation of normal hearing criteria
AUTHORS: Kelly Cristina Lira de Andrade, Thamyres Ataíde Bezerra Verçosa, Aline Tenório Lins Carnaúba, Pedro de Lemos Menezes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The present protocol aims to perform the screening of the subjects of the research investigating the auditory normality criteria. Thus, auditory symptoms, health history, medication use, exposure to noise and others are analyzed.</div></div>

[STEPS]
TITLE:  Operative Wound Care in Total Knee Arthroplasty
AUTHORS: Luisa Mululo, Fabricio Loures, Marcia Vanzillota, José Mauricio Moraes do Carmo, Marcelo Campos

[DESCRIPTION]
Guidelines for surgical wound care for patients undergoing total knee arthroplasty surgery performed at Hospital Universitario Pedro Ernesto. 

[STEPS]
TITLE: Anesthetic, analgesic and antibiotic protocol
AUTHORS: geoffrey.pages  

[DESCRIPTION]
This protocol was used to manage dogs with cruciate ligament disease treated by TPLO

[STEPS]
SECTION: Premedication (using acepromazine)
1. acepromazine (Calmivet; Vetoquinol™, Lure, France), 0.05 mg/kg intramuscularly (IM)
SECTION: Premedication
0.1. 
SECTION: Premedication (using diazepam)
2. diazepam (Valium; Roche™, Boulogne-Billancourt, France), 0.25 mg/kg intravenously (IV)
SECTION: Premedication (both)
3. morphine (Morphine; Lavoisier™, Paris, France), 0.2 mg/kg IV
SECTION: Induction
4. propofol (PropoVet; Zoetis™, Malakoff, France) 4 mg/kg IV, to effect
SECTION: Anesthetic agent
6. isoflurane (Isoflurin; Axience™, Pantin, France), 1.5-2.5%, in 100% oxygen
SECTION: Antibiotics
5. cefazolin (Cefazoline; Mylan™, Saint-Priest, France), 22 mg/kg IV 30 minutes before incision, repeated every 90 minutes until skin closure
SECTION: Postoperative medical management
7. morphine (Morphine; Lavoisier™, Paris, France), 0.2 mg/kg subcutaneously every 4 hours, until discharge
cefalexin (Rilexine; Virbac™, Carros, France), 15 mg/kg orally twice daily, for 5 days
local cryotherapy every 8 hours, until discharge
meloxicam (Metacam; Boehringer Ingelheim™, Reims, France), 0.1 mg/kg orally once daily, for 10 days
TITLE: Transforming pJC8 into HB101
AUTHORS: Bonnie Evans

[DESCRIPTION]
Taken from Mix and Go E. coli Transformation Kit (see attachment). 
Transforming E. coli HB101 with pJC8-empty cosmid to use as a control strain.

[STEPS]
SECTION: Before starting
1. Prepare tetracycline LB agar plates
 

SECTION: Before starting
2. Warm plates in 37 °C incubator

 

SECTION: Before starting
3. Prepare SOC medium
SECTION: Before starting
3.1. Get SOB medium and 20 % glucose from media kitchen
 
SECTION: Before starting
3.2. Add 2 mL 20 % glucose to 100 ml SOB medium
SECTION: Transformation
4. Isolate pJC8 cosmid from E. coli DH5α 
 

SECTION: Transformation
5. Thaw 100 uL aliquot of E. coli HB101 Mix & Go competent cells on ice
SECTION: Transformation
6. Add 5 uL pJC8 cosmid
SECTION: Transformation
7. Mix by tapping the tube and shaking downwards once
SECTION: Transformation
8. Place on ice for 5 minutes
SECTION: Transformation
9. Add 400 uL SOC medium
SECTION: Transformation
10. Incubate at 37 °C with shaking at 200 rpm for 1 hour

 

SECTION: Transformation
11. Pipette all (505 uL) of the transformation mix onto a tetracycline LB agar plate and spread using plating beads.
SECTION: Transformation
12. Dry under laminar flow for a few minutes
SECTION: Transformation
13. Incubate plate at 37 °C overnight
TITLE: Lumbar Puncture
AUTHORS: Clemens Scherzer, Bradley Hyman, Charles Jennings

[DESCRIPTION]
This protocol explains the Standard Operating Protocol for performing a Lumbar Puncture.

[BEFORE_START]
*Optimum time delay between withdrawal and freezing should be within 1-2 hours per European consensus. 

Ref: 
 


[GUIDELINES]
FREEZER STORAGE
  
Freezers are divided into 4 shelves, with 6 racks per shelf, and 24 boxes that can be held in each shelf. In total, 576 boxes, approximately 2,160 sample sets, can be stored in one -80°C freezer. The first three shelves are designated by visit number: Shelves A1-6 (top shelf) house samples from enrollment visits, shelves B1-6 (2nd shelf) house samples from the 1st year follow-up, and shelves C1-6 (3rd shelf) house samples from the 2nd year follow-up. Shelves D1-6 contain packed red blood cell tubes (PRBC), DNA, and RNA, extracted from blood as described in the protocols above. CSF is designated between two freezers in selected racks. Freezer storage and transactions of samples are recorded in the Freezerworks Inventory software.

[STEPS]
SECTION: Lumbar Puncture
1. Label the collection tubes with the sample ID as appropriate.
SECTION: Lumbar Puncture
2. Place aliquot tubes on dry ice prior to procedure so they are pre-cooled.
SECTION: Lumbar Puncture
3. Perform lumbar puncture using the atraumatic technique, inserting the needle with the bevel in parallel to the dura fibers. If bloody, discard the first 0.5 mL to clear any blood then collect. Record time of lumbar puncture.
SECTION: Lumbar Puncture
4. Collect 1 mL into one 14 mL polypropylene tube at Room temperature for routine diagnostic labs.

SECTION: Lumbar Puncture
5. Collect 20 mL into two 10 mL syringes at Room temperature. Then, transfer all 20 ml into one 50 ml Falcon tube, mix gently by inverting 3-4 times.
SECTION: Lumbar Puncture
6. Place 0.5 mL in one transfer tube for routine cell count, and 0.5 mL in another transfer tube for glucose and total protein. Send at Room temperature to core lab with completed requisition form.
 

SECTION: Lumbar Puncture
7. Spin at 400 x g, 10 min right after CSF is collected. Record time of spinning.

 


SECTION: Lumbar Puncture
8. Pipet 0.5 mL into the remainder (34-36 aliquots) 1.5 mL pre-cooled Fisherbrand silconized polypropylene transfer tubes.
SECTION: Lumbar Puncture
9. Freeze aliquots immediately on dry ice and transfer to Scherzer lab.
SECTION: Lumbar Puncture
10. Identify samples with obvious blood contamination.
SECTION: Lumbar Puncture
11. Scan specimens and place in -80°C freezer within 2 hours. Record time of freezing.

 

SECTION: Lumbar Puncture
12. Time of LP collection, spinning and freezer time will be recorded and entered into freezerworks.
SECTION: Lumbar Puncture
13. Specimens will be scanned, frozen, and stored in two separate freezers at the Scherzer lab.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocal characterized each focal fish (western mosquitofish, <em>Gambusia affinis</em>) for three standard indicators of personality using well-established experimental approaches: (1) boldness as latency to emerge from shelter and enter an unknown area, (2) activity in an open field tank and (3) sociability (i.e., shoaling tendencies), estimated as the time spent in the vicinity of a group of conspecifics. We tested each fish twice for its personality on two successive days, which allowed us to test for behavioral repeatability across both assessments.</p>

[BEFORE_START]
<p>We acclimated the fish to laboratory conditions for at least one month before we conducted behavioral experiments and maintained them in groups comprising both sexes at roughly even sex ratios, at densities of around 40 fish per tank, in several aerated and filtered 200-l aquaria under a 12:12 h light/dark regime. Aquaria were well equipped with plants, twigs and stones.</p>
<p>    We fed the fish twice a day <em>ad libitum</em> with commercially available flake food, frozen blood worms (chironomid larvae), as well as <em>Artemia salina</em> nauplii and shrimps. In the stock tanks and all experimental tanks, water temperature was kept at 25 ± 1 °C. Water quality was maintained by exchanging half of the water every two weeks, while one tenth of the water was exchanged every day in case of the (smaller) isolation tanks. Aged tap water was used for water changes and throughout the entire experiment.</p>
<p>    After acclimation period, we randomly selected adult focal fish from our stock tanks and isolated them, separated by sex, in 96-l tanks 24 hours prior to behavioral tests. To avoid aggressive interactions between individuals and to enable repeated testing of the same individuals, we kept each focal fish separately in 1.5-l transparent perforated plastic bottles, which allowed water exchange with the environment. Fish were returned to their isolation tanks between subsequent trials.</p>
<p>    A web cam (KC-QB960AK, Keeper, Shenzhen, China) was fixed in a central position approximately 70 cm above the test tank during all behavioral observations, allowing us to remotely observe the focal fish from above. We introduced an air stone connected to an air pump into the test tanks between trials to guarantee well-oxygenated water, and we changed the water every day after a testing session (every 4 trials). The test arena consisted of a glass tank (80 × 30 × 30 cm) that was filled with aged tap water to a height of 15 cm. The tank was placed on a gray plastic sheet with a fine white grid (5 cm squares). All outer sides were covered with black plastic foil to minimize disturbance.</p>

[GUIDELINES]
<p>This protocol describes preparation of personality assessment, boldness, activity and shoaling tendency measurement methods of <em>Gambusia affinis</em> under laboratory condition.</p>

[STEPS]
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>Adapted from the original publication     Rippka, R., DeRulles, J., Waterbury, J. B., Herdman, M. &amp; Stanier, R. Y. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. <em>Journal of General Microbiology</em> <strong>111</strong>, 1-61 (1979).</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Preparation of LRRK1 RCKW cryo-EM grids
AUTHORS: Mariusz  Matyszewski, David Snead

[DESCRIPTION]
Protocol used to create LRRK1 RCKW grids for cryo-EM used in Snead, Matyszewski, Dickey et al.

[BEFORE_START]
Decide which protein concentration to use, and create the proper LRRK1 buffers in order to obtain the right salt concentration (80 mM NaCl).

[STEPS]
SECTION: Freezing Grids
2. Plasma clean grids.
We used UltrAuFoil Holey Gold 1.2/1.3 300 mesh grids and plasma cleaned them in the Solarus II (Gatan) using the QuantiFoil Au preset.
SECTION: Freezing Grids
4. Apply protein to grids and plunge freeze.
We used a Vitrobot (FEI) to blot away excess sample and plunge freeze

 

SECTION: Freezing Grids
5. Store grids in liquid nitrogen until ready for imaging.
SECTION: Preparing Sample
1. Spin down purified LRRK1 RCKW. 10000 rcf, 10 min, 4 °C 
Leave protein on ice afterwards.

 

SECTION: Freezing Grids
3. Dilute samples to desired concentration in the LRRK1 buffer. Make sure final salt is at 80 mM NaCl.
For best results, make 10 µL samples, good for freezing 2 grids. This is to minimize time spent outside of storage buffer, reducing aggregation.
TITLE: 384 Well PicoGreen
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
Quant-iT™ PicoGreen® dsDNA Assay is used for detection and quantitation of double stranded DNA products. 

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]