eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This collection contains our main protocols for performing CITE-seq and Cell Hashing, specifically on <a href="http://mccarrolllab.com/dropseq/" target="_blank">Drop-seq</a> or <a href="https://www.10xgenomics.com/single-cell/" target="_blank">10x Genomics single cell 3′ v2 chemistry</a>. </p>
<p> </p>
<p><strong>CITE-seq: </strong></p>
<p> </p>
<p>Cellular Indexing of Transcriptomes and Epitopes by Sequencing (<a href="https://www.nature.com/articles/nmeth.4380" target="_blank">CITE-seq</a>) is a multimodal single cell phenotyping method developed in the <a href="http://www.nygenome.org/lab-groups-overview/technology-innovation-lab/" target="_blank">Technology Innovation lab</a> at the New York Genome Center in collaboration with the Satija lab.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/txwfw26.png" width="432" height="83" data-src="https://s3.amazonaws.com/pr-journal/txvfw26.png" data-ofn="Screen Shot 2018-02-26 at 5.42.11 PM.png" /></p>
<p> </p>
<p>CITE-seq uses DNA-barcoded antibodies to convert detection of proteins into a quantitative, sequenceable readout.  Antibody-bound oligos act as synthetic transcripts that are captured during most large-scale oligodT-based scRNA-seq library preparation protocols (e.g. 10x Genomics, Drop-seq, ddSeq).</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/txyfw26.png" width="579" height="398" data-src="https://s3.amazonaws.com/pr-journal/txxfw26.png" data-ofn="Screen Shot 2018-02-26 at 5.42.29 PM.png" /></p>
<p> </p>
<p>This allows for immunophenotyping of cells with a potentially limitless number of markers and unbiased transcriptome analysis using existing single-cell sequencing approaches.</p>
<p> </p>
<p><strong>Cell Hashing:</strong></p>
<p> </p>
<p>Sample multiplexing and super-loading on single cell RNA-sequencing platforms.</p>
<p> </p>
<p><a href="https://www.biorxiv.org/content/early/2017/12/21/237693" target="_blank">Cell Hashing</a> uses a series of oligo-tagged antibodies against ubiquitously expressed surface proteins with different barcodes to uniquely label cells from distinct samples, which can be subsequently pooled in one scRNA-seq run. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its sample of origin, and robustly identify doublets originating from multiple samples.</p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/tx2fw26.png" width="578" height="235" data-src="https://s3.amazonaws.com/pr-journal/txzfw26.png" data-ofn="Screen Shot 2018-02-26 at 5.44.55 PM.png" /></p>
<p> </p>
<p> </p>

[STEPS]
TITLE: NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II End Repair/dA Tailing Module (NEB #E7546)
AUTHORS: jbonnevie

[DESCRIPTION]
This module is part of the Ultra™ II workflow, and is optimized for use with the NEBNext®Ultra II End Repair/dA-Tailing Module (NEB #E7546), for Illumina®-compatible library construction.

The NEBNext Ultra II Ligation Module is optimized for use with the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546) or the NEBNext Ultra II FS DNA Module (NEB #E7810).

[BEFORE_START]
Starting Material:
500 pg–1 µg fragmented DNA that has been end repaired and dA-Tailed using the NEBNext End Repair/dA-Tailing Module (NEB #E7546).
 


[GUIDELINES]
Safe Stop Point: This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol. 

Caution: Signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.

Color: A color listed before or after a reagent name indicates the cap color of the reagent to be added.

Adaptor Dilution Guidelines

The appropriate adaptor dilution for your sample input and type may need to be optimized experimentally. The dilutions provided here are a general starting point.

Table 1.1: Adaptor Dilution 
 
 

 

[STEPS]
SECTION: Ligation/End Prep
1. Add the following products directly to the End Prep Reaction Mixture:
 
* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.
** The NEBNext adaptor is provided in the NEBNext Oligo kit options, which can be found at www.neb.com/oligos.
 

 

 

 

SECTION: Ligation/End Prep
2. Set a 100 µl or 200 µl pipette to 80 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. 
 

SECTION: Ligation/End Prep
3. Incubate at 20 °C for 15 min in a thermal cycler with the heated lid off. 




SECTION: Ligation/End Prep
4. Add 3 µl of (red) USER® Enzyme to the ligation mixture from Step 3. 
 

 

SECTION: Ligation/End Prep
5. Mix well and incubate at 37 °C for 15 min with the heated lid set to ≥ 47 °C.
SECTION: Ligation/End Prep
6. DNA is now ready for size selection or cleanup.
TITLE: Biochemical Measures of Neuropathy - (H2) DCFDA
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/biu84kcn.jpg" /></div></div>

[STEPS]
?. [Assay Preparation:]
Set up plate layout in AscentFL software. Choose DCFDA.sed and fill in your layout. Save your file as DCxxxxxx.sed with xxxxxx being the date in yy/mm/dd format.
?. [Assay Preparation:]
Treat cells per experimental paradigm.
?. [Assay Preparation:]
15-30 minutes prior to reading, add 3.23 µL of DCFDA stock to 15 mL media without serum.
?. [Assay Preparation:]
Rinse cells with HBSS.
?. [Assay Preparation:]
Place plate into Fluroskan holder and click START.
?. [Assay Preparation:]
Take readings using 485 nm ex, 520 nm em filter pair.
?. [Assay Preparation:]
Save sheet with .xls extension into the DCFDA data folder or your own folder. Use the naming convention DCXXXXXX.xls, where XXXX is the date in yymmdd format.
TITLE: Mouse Lung Digestion
AUTHORS: gagandeep kaur, Irfan Rahman

[DESCRIPTION]
The objective of this protocol is to digest the mouse lungs to perform scRNA analyses

[STEPS]
SECTION: Lung Tissue Dissociation
2. 
Immediately transfer the tubes to MACS Tissue Dissociator and run user-defined program named m_lung_01_02 for mouse lungs and m_heart_01.01 for mouse heart.

SECTION: Lung Tissue Dissociation
1. 1)    Mince tissue finely, place into 50ml GentleMACS C-tube with liberase enzymatic cocktail  a.     For 50mg tissue use 0.5 ml liberase + 2ml DMEM + 2ul 5 unit/ul DNase I)
SECTION: Lung Tissue Dissociation
3. Thereafter, place 50ml conical with digestion into an incubating rocker for 30 minutes at 37 degrees C. 
NOTE: If you still see chunks of lung tissue in the tubes after this step then transfer to the MACS Tissue Dissociator and run user-defined program named m_lung_02_01 for mouse lungs for 10-15 sec.
SECTION: Lung Tissue Dissociation
4. Remove conical from rocker and strain the cell suspension through 70 micron into 50mL tube 
SECTION: Lung Tissue Dissociation
5. Add 3 ml DMEM (10% FBS) through the strainer to collect the remaining cell suspension
SECTION: Lung Tissue Dissociation
6. After straining, centrifuge at 300g for 5 minutes at 4degree C.
SECTION: Lung Tissue Dissociation
7. Remove supernatant, add 500 µl of red blood cell lysis to cell pellet. Incubate the cell suspension on ice for 5 min.
SECTION: Lung Tissue Dissociation
8. After incubation add 4.5 mL of DMEM (10% FBS) to the suspension and centrifuge at 300g for 5 minutes at 4degree C.
SECTION: Lung Tissue Dissociation
9. Remove supernatant, re-suspend pellet in 2 mL DMEM (10%FBS)
SECTION: Lung Tissue Dissociation
10. Count cells and viability using AO/PI method, confirm reading under microscope
SECTION: Lung Tissue Dissociation
11. Submitted the digested suspension to the Genomic Research Center for scRNA seq analyses
TITLE: Traditional Chinese Medicine as add-on treatment of triple/quadruple therapy for Helicobacter pylori infection: an overview of systematic reviews and meta-analyses (Protocol)
AUTHORS: Xiaobei Si, Shuai Wang, Chun-Lian Lai, De-Ying Bi, Yu Lan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Background:</span><span>In recent years, many studies reported the anti-Hp effects of Traditional Chinese Medicine. Based on these studies, there had been many relevant systemic reviews and meta analyses with variable results</span></div><div class = "text-block"><span style = "font-style:italic;">Aim: </span><span>This overview aims to investigate the general characteristics and assess the methodological quality of these systemic reviews/meta ana lyses (SR/MAs).</span></div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Method:</span><span> In this overview, we intend to includ systemic reviews/meta analyses with comparison of Chinese medicine and western medicine on helicobacter pylori infection by searching databases of Medline, Embase, Web of Science, China National Knowledge Infrastructure Database (CNKI), Chinese VIP Information (VIP), Chinese Medical Databases (CMB) and Wan-Fang Database until December 16th, 2018. AMSTAR and GRADE tools will be adopted to assess the methodological quality and evidence quality characteristics. </span></div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Conclusion:</span><span>The methodological quality of these SR/MAs will be assessed as clinical evidence and our result may help further use of them. </span></div></div>

[STEPS]
TITLE: Latex beads behavior assay
AUTHORS: Jorge Fernández

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for testing latex beads agreggation after resuspension in different buffers. It allows determining how beads storage conditions can influence their ability to move freely trhough a membrane.</div></div>

[STEPS]
?. [Latex Beads Resuspension]
Centrifugate 4 different aliquots of 40 μL 2.5 %wt. latex bead stock suspension at 15.000 rpm for 3 minutes. After centrifugation remove the supernatat slowly.
?. [Latex Beads Resuspension]
Resuspend the beads in 100 μL of the four different buffers: distilled water, PBS-T, Bicarbonate Buffer and MES buffer. For resuspending beads repeated pipetting it's highly recommended, aspiring and blowing out in the eppendorf tube.Label the different eppendorfs with the buffer added.
?. [Latex Beads Migration]
For beads migration test, wait 10 minutes and pipette 10μL of each bead suspension on a piece of nitrocellulose membrane.  Separate each drop one to each other at least 8 mm.
?. [Latex Beads Migration]
Wait until the deposited sample has migrated completely, and circle on the plastic backing of the nitrocellulose membrane the outline of the sample eluted front. Vissible results of beads retention in the membrane should be vissible at this point.
?. [Latex beads reestablishment]
Repeat step 1 and 2, resuspending all the beads in distilled water.
?. [Latex beads reestablishment]
Repeat step 3-4 with the resuspended bead stocks.
TITLE: Analysing mitochondrial oxygen consumption using the Seahorse XFe96 analyzer
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for analysing mitochondrial oxygen consumption using the Seahorse XFe96 analyzer

[STEPS]
SECTION: Day 1
1. Seed cells in 10 cm plates aiming for a confluency of ~80-90% at the time of treatment 
SECTION: Day 1
2. Add 200 µL of Seahorse XFe calibrant solution to each well of a Seahorse (Agilent) cartridge plate, and incubate   at 37 °C in a CO2-free incubator

SECTION: Day 2
3. Isolate mitochondria (see previously published protocol) with the following modifications: cells were scraped into ice cold modified isolation buffer (70 mM sucrose, 210 mM mannitol, 1 mM EGTA, 0.5% w/v BSA (fatty acid free), 5 mM HEPES pH 7.2), cell pellets were stored on ice prior to homogenization, and mitochondrial samples were stored on ice and immediately assayed after quantification 
SECTION: Day 2
4. Quantify mitochondria by bicinchoninic assay
SECTION: Day 2
5. Aliquot out 15 µgof mitochondria per sample, diluting each aliquot to a final volume of 25 µL in mitochondrial assay solution (MAS: 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl26H2O, 1 mM EGTA, 0.1% w/v BSA (fatty acid free), 2 mM HEPES pH 7.2), and leave the samples on ice until needed 

SECTION: Day 2
6. Pre-chill a Seahorse sample plate on ice
SECTION: Day 2
7. Make up the substrate solution (10 mM Glutamate, 10 mM malate in MAS buffer) and place at 37 °C in a CO2 free incubator for at least60 min

SECTION: Day 2
8. To the equilibrated cartridge plate, load 20 µL of 20 mM ADP, 22 µL50 ug/uL oligomycin, 24 µL of 10 uM FCCP, 26 µL of 40 uM of Antimycin A into the corresponding ports of each well

SECTION: Day 2
9. Incubate the cartridge at 37 °Cin a CO2-free incubator for 45 min, and begin the calibration sequence on the Seahorse XFe96 analyser so that it’s completion corresponds with step 12 (takes ~30 min). 

SECTION: Day 2
11. Centrifuge the plate at 2000 rcf, 20 min, 4 °C, and place the plate on ice until required 



SECTION: Day 2
10. During step 8, add a 25 µL aliquot of mitochondria to each corresponding well of the prechilled Seahorse sample plate

SECTION: Day 2
12. Add 155 µLof pre-warmed substrate solution to each sample well 

SECTION: Day 2
13.  Eject the calibration plate from the analyser and replace it with the sample plate
SECTION: Day 2
14. Run the following protocol on the analyser: 
Basal (3 min mix, 3 min measure, 3 min mix, 3 min measure)
ADP (injection, 30 sec mix, 3 min measure)
Oligomycin (injection, 30 sec mix, 30 sec wait, 3 min measure)
FCCP (injection, 20 sec mix, 3 min measure)
Antimycin A (injection, 30 sec mix, 3 min measure)
SECTION: Day 2
15. If analysing mitochondrial respiration across multiple days, perform step 2 of day 1 the day before the time point analysis, and perform step 3 – 14 on each day of the time course with the appropriate vehicle controls each day.
TITLE: 16s rDNA gene amplification protocol
AUTHORS: Nataly Ruiz Quinones, Cristian Daniel Asmat Ortega, Rene Flores Clavo

[STEPS]
?. [Master mix preparation]
In a polypropylene tube, mix the following reagents for the final volume of 25 μL per reaction
?. [Master mix preparation]
Add 2.5 μL of 10X Taq buffer (final concentration 1X)
?. [Master mix preparation]
Add 0.75 μL of MgCl2 (50 mM) (final concentration 2.5 mM)
?. [Master mix preparation]
Add 0.2 μL dNTP (2.5 mM each dNTP) (final concentration 0,02 mM)
?. [Master mix preparation]
Add 0.5 μL of each primer (20 μM ) (final concentration 0,4 μM)Primer 10f  5’-GAG TTT GAT CCT GGC TCA G-3’ Primer 1401r 5’-CGG  TGT GTA CAA GGC CCG GGA ACG-3’ or 1492r 5’- ACC TTG TTA CGA CTT-3’  orPrimer 1492r 5’- ACC TTG TTA CGA CTT-3’
?. [Master mix preparation]
Add 5.0 μL of the DNA template (50-100 ng/μL) (final concentration at 1-2,5 ng per reaction)
?. [Master mix preparation]
Add sterile deionized water until filling at 25 μL per reaction.
?. [PCR reaction]
For PCR amplification, use the following cycling program
?. [PCR reaction]
Run an Initial denaturation step of 5 min at 95°C
?. [PCR reaction]
Run 30 cycles of 1 min at 94°C for denaturation, 1 min at 55°C for annealing, 3 min at 72°C for extension
?. [PCR reaction]
Run a 3 min at 72°C for the final extension
?. [PCR reaction]
Store amplification products at 4°C until visualization.
?. [Visualization]
To analyze and visualize the amplified products, put 2 μL of the PCR product with 2 μL of loading buffer dye in agarose gel electrophoresis with 1X TBE stained with SYBER safe at 1:10000.
?. [Master mix preparation]
Add 0,2 μL of Taq DNA polymerase (5 U/μL) (Final concentration 0,04 U)
?. [Visualization]
To compare, add 1 μL of 1kb DNA ladder (final concentration 0,5 ng/μL) and observe a band of 1500 pb approximately.
TITLE: CTAB DNA Extraction Protocol for Mollusks
AUTHORS: Lavanya M Vythalingam, Tim Regan, Tim Bean, Carolina Peñaloza

[DESCRIPTION]
This is a comprehensive CTAB extraction protocol intended for DNA extraction of mollusks. It includes detailed methodology and several reagent preparation.

[STEPS]
SECTION: Method
1. 
Slice tissue sample using a clean, sterile scalpel. Put sample into a 1.5 ml tube with  400 µL  .

SECTION: Method
2. 
Add 10 µL   20 mg/mL and invert tube to mix.

SECTION: Method
3. 
Incubate at 50 °C for 60 minutes until lysed (or longer depending on tissue thickness). Invert tube occasionally to mix sample.

SECTION: Method
4. Add 400 µL   . Gently shake tube to emulsify sample.

SECTION: Method
5. 
Leave at room temperature for 2 minutes and shake tube to mix.
SECTION: Method
6. 
Spin tube at 13 000 rpm for 2 minutes and transfer upper layer to a 1.5 ml tube. 
SECTION: Method
8. 
Add 1 equal volume of  .

SECTION: Method
9. 
Spin tube at 13 000 rpm for 2 minutes and transfer upper layer to a new 1.5 ml tube containing 900 µL CTAB dilution solution. Gently invert tube to mix. Leave in fridge overnight (optional).

SECTION: Method
10.  
Spin tube at 13 000 rpm for 10 minutes.
SECTION: Method
11. 
Discard supernatant with a micropipette and add 1000 µL 0.4 M NaCl in TE. Invert tube to wash.

SECTION: Method
12. 
Spin tube at 13 000 rpm for 5 minutes and remove supernatant.
SECTION: Method
13. 
Add 300 µL 1.42 M NaCl in TE to dissociate the CTAB from the DNA. Invert tube until pellet becomes transparent. 
 


SECTION: Method
16. 
Resuspend in 50 µL EB buffer. Use lesser volume of EB buffer if a higher concentration is required. 

SECTION: Reagent preparation
20. 
CTAB Extraction Solution
 

SECTION: Reagent preparation
20.1. 
CTAB Dilution Solution


 

SECTION: Reagent preparation
20.2. 
0.4M Nacl in TE
 

SECTION: Reagent preparation
20.3. 
1.42 M NaCl in TE
 

SECTION: Notes
18. 
The Nucleic acid quality is measured using a Nanodrop, and the A260/A230 ratio of 1.8-2.0 indicates good DNA purity. Qubit can be used to determine nucleic acid concentration.
SECTION: Method
7. Add 5 µL of  10 mg/mL . Leave for 30 minutes at 37 °C .

SECTION: Notes
19. 
Store DNA sample at  -20°C until further use.
SECTION: Method
14. 
Add 600 µL   (previously left at -20 °C ). Invert manually until precipitation appears.

 

SECTION: Notes
17. 
All centrifugations were performed using a benchtop centrifuge (Eppendorf - 5415 D)



SECTION: Method
15. 
Centrifuge at full speed for 8 minutes, then remove supernatant. Dry at room temperature or in heat block at 37 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocoll outlining the Preperation and transformation of electro-competent cells for <em>Vibrio natriegens</em></p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>Preparation of electrocompetent cells (<em>Vibrio natrigens</em>)</p>
<p> </p>
<ol>
<li>10 mL BHI (Brain haert infusion) + v2 salts Overnight culture</li>
<li>Inoculation of a new BHI + v2 media with 1% of the overnight culture as inoculum.</li>
<li>Grow at 37°C shaking to an OD of 0.5</li>
<li>Chill the culture on ice for 15 min</li>
<li>Use a chilled (4°C) centrifuge at 4,500 r.p.m. for 20 min</li>
<li>Decant the supernatant diligently and carefully</li>
<li>Resuspend gently in 5-10 mL Electroporation-Buffer (680 mM sucrose, 7 mM K2HPO4, pH 7), then fill the falcon tube to the top.</li>
<li>To wash, spin again at 4.500 r.p.m. for 15 min at 4 °C, then resuspend gently in 5 mL Electroporation-Buffer. Repeat washing twice for a total of three times.</li>
<li>Spin again at 4.500 r.p.m. for 15 min at 4 °C, decant supernatant and resuspend carefully in the residual buffer.</li>
<li>Adjust volume for a OD of 16</li>
<li>Aliquot in chilled Micro-Reaction-Tubes, then snap freeze in liquid nitrogen</li>
<li>Store at –80°C</li>
</ol>
<p><br /> Electroporation</p>
<p> </p>
<ol>
<li>remove one aliquot with electrocompetent cells from storage</li>
<li>keep on ice until thawed</li>
<li>add plasmid and mix gently</li>
<li>Transfer to chilled electroporation cuvette</li>
<li>Electroporation is done with the following parameters: <strong>700</strong>(–900) V, 25 μF, 200 Ω  in a 1mm cuvette</li>
<li>Recover for one hour at 37°C in brain heart infusion with v2 salts</li>
<li>Plate on LB2 agar plates. </li>
</ol>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Processing wastewater samples for bacterial &amp; viral targets enrichment 
AUTHORS: Victor Mabasa

[DESCRIPTION]
This wastewater sample processing protocol by the Wastewater Genomics Syndicate at the National Institute of Communicable Diseases (NICD) is designed to enrich bacteria and viruses from the settled solids and supernatant components of wastewater, as different pathogens partition differently within this matrix. The end products are waterless solids and clarified wastewater supernatants, ready for downstream applications such as ultrafiltration, bead enrichment and nucleic acid extraction.   

[STEPS]
SECTION: Sample Collection &amp; Handling
1. Using correct personal protective equipment (PPE), collect a 1 000 mL grab wastewater sample from each location in a sterile bottle and transfer them to the laboratory in a cold chain (keep in a lab fridge at 4°C). it is important to proceed to the next step within the next 23 hours after sample collection to prevent major degradation of microorganisms. 
SECTION: Preparing for centrifugation
2. All work should be performed in a Class II biological safety cabinet while wearing appropriate PPE. Clean your workspace with a 1% Virkon solution and a 70% ethanol solution. Double-line your waste bucket with biohazard waste plastic, and ensure all your filter tips are refilled. Finally, label 250 mL conical centrifuge bottles, one for each wastewater sample.
SECTION: Centrifugation
3. Transfer 250 mL of the wastewater samples to the corresponding conical tubes

SECTION: Centrifugation
4. Centrifuge at 4 650 × g (deceleration speed set at 5), at 4oC for 10 minutes.
SECTION: Centrifugation
5. Carefully transfer the supernatant from the 250 mL conical bottle to a clean, marked 1 000 mL bottle without disturbing the settled solids.  
SECTION: Centrifugation
6. Repeat steps 3 to 5 until the original sample is finished. 
SECTION: Centrifugation
7. Keep the clarified wastewater sample at 4oC for different downstream applications (check our page for different nucleic acid extraction protocols from clarified wastewater). 
SECTION: Transfer settled solids
8. Use clean, plain wooden sticks to pick out settled solids and pack them into marked 2 mL cryo-tubes. Centrifuge briefly to remove water.  
SECTION: Transfer settled solids
9. Store the settled solids in the –80°C freezer until needed for downstream applications. (check our page for different nucleic acid extraction protocols from settled solids).