eamag/protocols-with-steps huggingface dataset:
TITLE: Quality control analysis for 10X snRNA-seq 
AUTHORS: Daniel  Jacobsen, Dinh H Diep

[DESCRIPTION]
Here we describe a computational protocol for performing quality control analysis on shallow sequencing data obtained from 10X snRNA-seq experiments. The workflow starts with raw MiSeq run folders and uses cellranger to generate count matrices. The raw count matrices are analyzed and sequencing saturation plots are generated. The saturation plots are then compared against plots from a reference set of libraries with varying qualities (bad, fair, good, great), thus allowing for the determination of sequencing requirements as well as an assessment of the overall quality of each 10X snRNA experiment.

[STEPS]
1. Install cellranger using instructions from https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/installation

 

4. Install anaconda or miniconda Python distributions following given instructions.
Get anaconda from here: https://www.anaconda.com/products/distribution , OR
get miniconda from here: https://docs.conda.io/en/latest/miniconda.html
6. Use conda to install bcl2fastq with the following terminal command:

 

7. Use conda to install required python packages with the following terminal command:

 

5. Install preseq using given instructions from http://smithlabresearch.org/software/preseq/.


 

8. Run cellranger mkfastq to generate fastq files. Make sure that the following placeholders are set to the correct paths and desired names.
<FASTQ_OUT> is the name of the output folder
<RUN> is the path to the MiSeq run folder
<CSV> is the path to the sample-sheet.csv file

 

9. Run cellranger count. Make sure that the following placeholders are set to the correct paths and desired names.
<ID> is the name of the output folder for the sample
<SAMPLE> is the sample name used for the sample in the sample-sheet.csv file
<REF> is the path to the cellranger reference data folder
<NUM> is the number of expected cells from the experiment


 


10. Run the preseq script in the folder downloaded from this protocol. Make sure that the following placeholders are sset to the correct paths and names.

<PATH_TO_FOLDER> is the path to the folder that was extracted from the tar.gz file.
<ID> is the name of the output folder for the sample generated with cellranger.
 

2. Download the tar.gz file from this protocol.
3. Extract the tar.gz file from this protocol.
<FILENAME> is the name of the downloaded tar.gz file.
 

11. View outputs.

<ID>.lc_extrap_log.txt contains preseq statistics
<ID>.lc_extrap_output.png to view the sequencing saturation plots
<ID>/outs/web_summary.html to view the cellranger analyses
<ID>/outs/summary.csv to view quality statistics generated by cellranger
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This procedure is optimized for the isolation of 10<sup>7</sup> to 2 x 10<sup>8</sup> cells per tube from human peripheral blood mononuclear cells (PBMCs). If working with fewer cells keep volumes as indicated for 10<sup>7</sup> cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. To maximize the purity of CD4+ T cells we recommend the depletion of monocytes, as human monocytes express CD4. To deplete monocytes from human PBMCs, we recommend the use of MojoSort™ Human CD14 Selection Kit (Cat. No. 480025/480026). Alternatively, you may consider the Human CD4 T Cell Selection Kit (Cat. No. 480038). After monocyte depletion proceed to isolate the CD4+ T cells. You can also use other methods to deplete monocytes. If you wish to include monocytes in your CD4+ cell isolation, depletion is not required.</p>

[GUIDELINES]
<p><strong>Product description and procedure summary:<br /></strong>The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>
<p> </p>
<p><strong>Reagents and instruments required:</strong></p>
<p><br />MojoSort™ Buffer (5X) (Cat. No. 480017)<br />MojoSort™ Magnet (Cat. No. 480019) or compatible magnetic separation system<br />Adjustable pipettes<br />70 μm filters (one per sample)<br />5 mL (12 x 75 mm) polystyrene tubes<br />Reagents for sample preparation<br />Reagents and instruments (Flow cytometer) to determine yield and purity</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dr9ecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dr8ecje.jpg" data-ofn="mojosort human cd4 nano.JPG" /></p>
<p><strong>Application notes</strong>: To use this product in magnetic separation columns, a titration of the Nanobeads should be performed. Optimal concentration for magnetic separation columns is lot-specific.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?.
TITLE: JMN-MSMP Muscle Single Cell Isolation
AUTHORS: ccherry

[DESCRIPTION]
Single cell isolation from muscle

[STEPS]
SECTION: Single cell isolation
1. Tissue was finely diced in RPMI 1640 (Gibco) with 1.6 Wunsh U/mL Liberase TL (Roche Diagnostics) and 0.2 mgl/mL deoxyribonuclease I (Roche Diagnostics).
SECTION: Single cell isolation
2. Finely diced tissue was incubated at 37C for 45 minutes.
SECTION: Single cell isolation
3. Digested tissues were ground subsequently through 100um and 70um strainers (Thermo Fisher Scientific).
SECTION: Single cell isolation
4. Cells were centrifuged at 300g for 5 minutes and washed twice with 1x PBS
TITLE: Cell treatments for time-lapse experiments
AUTHORS: Electra Brunialti, Alessandro Maria Villa, Paolo Ciana

[DESCRIPTION]
Cell treatments for time-lapse experiments.

[STEPS]
1. For the timelapse experiments, cell cultures were treated for 6 h with vehicle (water) or lipopolysaccharide (LPS) at the final concentration of 10 µg/ml LPS O111:B4, (Cat. L2630, Merk) starting from a 0,5 mg/ml stock solution, and then subjected to timelapse microscopy for 2h. For the GCase inhibition, cells were treated for 48 h with 200 µM CBE (Cat. 234599, Merk) starting from a 10 mM stock solution, or vehicle (water), and then subjected to timelapse microscopy for 2h.
TITLE: LEGACY01: ADVERSE EVENTS
AUTHORS: Katrina M Pollock, Calliope Dendrou

[DESCRIPTION]
This protocol details about adverse events in an experimental medicine study of seasonal influenza vaccination responses in Lymph nodE single-cell Genomics in AnCestrY (LEGACY01).

[GUIDELINES]
DEFINITIONS

Adverse Event (AE): any untoward medical occurrence in a patient or clinical study subject.

Serious Adverse Event (SAE): any untoward medical occurrence or effect that:

Results in death

Is life-threatening – refers to an event in which the subject was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it were more severe

Requires hospitalisation, or prolongation of existing inpatients’ hospitalisation

Results in persistent or significant disability or incapacity

Is a congenital anomaly or birth defect

Medical judgement should be exercised in deciding whether an AE is serious in other situations. Important AEs that are not immediately life-threatening or do not result in death or hospitalisation but may jeopardise the subject or may require intervention to prevent one of the other outcomes listed in the definition above, should also be considered serious.

REPORTING PROCEDURES

Adverse events should be reported depending on the nature of the event and the reporting procedures below should be followed. Any questions concerning adverse event reporting should be directed to the Chief Investigator in the first instance. Events should be graded according to the FDA: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials, taking account of local laboratory reference ranges. https://www.fda.gov/media/73679/download

NON-SERIOUS AES

These will be recorded in the following instances:
Those occurring in the 5 days following lymph node tissue sampling 
Those occurring in the 28 days following influenza vaccination 

SERIOUS AES

SAEs must be recorded during the entire study.

An SAE form should be completed and emailed to the Chief Investigator within 1440 min. However, hospitalisations for elective treatment of a pre-existing condition do not need reporting as SAEs.

All SAEs should be reported to the REC which gave favourable opinion of the study, where in the opinion of the Chief Investigator the event was:
‘related’, i.e. resulted from the administration of any of the research procedures; and
‘unexpected’, i.e. an event that is not listed in the protocol as an expected occurrence

Reports of related and unexpected SAEs should be submitted within 15 days of the Chief Investigator becoming aware of the event, using the NRES SAE form for non-IMP studies. The Chief Investigator must also notify the Sponsor of all related and unexpected SAEs.

The aQIV is subject to additional monitoring. At the Chief Investigator's discretion, side effects of vaccination that are suspected adverse reactions will be reported to the MHRA via its Yellow Card Scheme: www.mhra.gov/yellowcard

 


[STEPS]
TITLE: Immunofluorescence Staining and Analysis of Astrocyte-Neuron Co-Cultures
AUTHORS: Shiyi Wang

[DESCRIPTION]
Immunofluorescence Staining and Analysis of Astrocyte-Neuron Co-Cultures

[STEPS]
1.  **Fixation**
1.1. - Fix astrocyte-neuron co-cultures on glass coverslips on Day in Vitro 12 (DIV 12) with warm 4% paraformaldehyde (PFA) for 7 minutes.
1.2. - Wash coverslips 3 times with phosphate-buffered saline (PBS).
2.  **Blocking**
2.1. - Block coverslips in a blocking buffer containing 50% normal goat serum (NGS) and 0.4% Triton X-100 for 30 minutes at room temperature.
2.2. - Wash coverslips with PBS.
3. **Primary Antibody Incubation**
3.1. - Incubate samples overnight at 4°C in primary antibodies diluted in blocking buffer containing 10% NGS.
3.2. - Incubate coverslips in Alexa Fluor conjugated secondary antibodies (Life Technologies) for 2 hours at room temperature.
3.3. - Wash coverslips again with PBS.
4. **Mounting**
4.1. - Mount coverslips onto glass slides (VWR Scientific) using Vectashield mounting media containing DAPI (Vector Labs).
4.2. - Seal coverslips with nail polish.
5. **Imaging**
5.1. - Image coverslips using an AxioImager M1 fluorescence microscope (Zeiss) at 40x magnification in red, green, and/or DAPI channels using a CCD camera.
6. **Morphological Analysis**
6.1. - Analyze astrocyte morphological complexity using FIJI with the Sholl analysis plugin (https://github.com/Eroglu-Lab/In-Vitro-Sholl).
6.2. - Ensure analysis is performed on healthy astrocytes with strong expression of fluorescent markers and single, non-overlapping nuclei (DAPI stain).
7. **Statistical Analysis**
7.1. - Conduct statistical analyses using custom code in R (https://github.com/Eroglu-Lab/In-Vitro-Sholl).
7.2. - Use a mixed-effect model with Tukey post-test for Sholl analysis to evaluate differences between experimental conditions, treating variability per experiment as a random effect.
8. **Quality Control**
8.1. - Verify the health of astrocyte-neuron co-cultures by ensuring the peak number of astrocyte intersections is ≥ 25 in the control condition.
8.2. - Document the exact number of independent experiments and cells analyzed in the figure legend for each experiment.
9. Notes:
9.1. - Maintain sterility and avoid contamination during all steps.
9.2. - Ensure imaging settings are consistent across experimental conditions for accurate comparison.
9.3. - Validate antibody specificity and optimize staining conditions for reproducibility.
TITLE: Lysis of diatom bulk samples
AUTHORS: Till Macher

[DESCRIPTION]
This protocol describes the steps of sample preparation and lysis before DNA extraction for the diatoms metabarcoding protocol of the LeeseLab. The protocol was established in the GeDNA project.


[GUIDELINES]
Reagents are potentially damaging to the environment. Dispose waste responsibly.

[STEPS]
SECTION: Sample concentration
7. Centrifuge 50 mL falcon tubes containing the diatom samples 4000 x g, 10 min 

SECTION: Sample concentration
8. Discard supernatant EtOH (can be poured from the falcon tube).
SECTION: Sample concentration
9. Add 10 mL to each sample (in falcon tube). Vortex samples to elute pellets again.
SECTION: Sample lysis
10. Prepare each one 2 mL twist-top tube with each one spoon of 2.0 mm (ca. 10) and 1.0 mm (ca. 30) Zirconia beads. Label tubes both on the side and the lid.
SECTION: Sample lysis
11. Add 1 mL to each twist-top tube.
 

SECTION: Sample lysis
12. Centrifuge samples  6000 x g, 5 min .

SECTION: Sample lysis
13. Discard supernatant EtOH (use 1000 µL tips).
SECTION: Sample lysis
14. Add 900 µL and 100 µL to each sample.


 

SECTION: Sample lysis
15. Bead-beat for 2 min at   .

SECTION: Sample lysis
16. Incubate samples at 55 °C for 20 min at   .

SECTION: Sample lysis
17. Store samples at -20 °C until DNA extraction.

SECTION: Sample collection (short description)
1. Perform multi-habitat diatoms sampling (depending on the protocol).
SECTION: Sample collection (short description)
2. Prepare one sterile 50 mL Falcon tube for each sample. Label the Falcon tube with EtOH resistant labels (e.g. printed labels). 
SECTION: Sample collection (short description)
3. Add 10 mL (e.g. scrape sample) to the Falcon tube.

SECTION: Sample collection (short description)
4. Add 40 mL to the Falcon tube.

SECTION: Sample collection (short description)
5. Mix sample by inverting the Falcon tube.
SECTION: Sample collection (short description)
6. Store samples under dark conditions at Room temperature or at 4 °C until lysis.
TITLE: RNA Isolation
AUTHORS: Natalie Brown

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Real Time Polymerase Chain Reaction</div></div>

[STEPS]
?. ●	Get plates, aspirate media
?. ●	Add 2mL ice cold DPBS
?. ○	Aspirate DPBS
?. ●	Add 500uL Trizol/well in chemical hood
?. ●	Bring yellow tubes to hood, collect trizol and add to tubes
?. ●	Add 100uL chloroform to tubes
?. ○	hand shake 15s
?. ○	Place on ice 5min until see layers (aq/lipids/bottom)
?. ●	Spin full speed 20min cold centrifuge
?. ○	During, make 70% ethanol on ice (3mL RNAse-free water + 7mL ethanol)
?. ○	Make new ep set 1-9
?. ●	In hood, collect top layer after spinning and place into eppendorfs
?. ○	**take note of first volume and make all the same
?. ○	Mix 1:1 ethanol with supernatant, use same tip to transfer from eppendorfs into pink spin tubes
?. ●	Spin 9000x 1min bench centrifuge
?. ○	Discard flowthrough in hood, keep tube
?. ●	Add 350uL RW1 buffer to tubes
?. ○	Spin 1 min
?. ○	During, prepare DNAse soln (with syringe)
?. ●	Add 70uL DNAse soln to middle of each column, leave 15min RT
?. ●	Repeat RW1 and spin
?. ○	Discard collection tube and get new
?. ●	Wash RPE 500uL
?. ○	Spin 1min
?. ○	Discard collection and keep tubes
?. ●	Repeat RPE, spin 2min
?. ○	Discard tubes
?. ●	Spin 1min empty to dry
?. ○	Get new eppendorfs, label
?. ●	Place pink tubes on top of eppendorfs
?. ●	Add 30uL RNAse free water, centrifuge 1min
?. ○	Close tubes as quickly as possible and keep on ice→ nanodrop
TITLE: Protein purification strep-tag on gravity column 
AUTHORS: Cleo B.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein purification of a protein with a strep-tag on a gravity column. </div></div>

[STEPS]
?. [Protein extraction]
Place post-induction culture from Protein expression using E. coli strain BL21DE3 on ice
?. [Protein extraction]
Centrifuge culture at 5000xg for at
4 °C
?. [Protein extraction]
The pellet is resuspended in  buffer W per cell culture containing one crushed cOmplete mini tablet.
1 ml
100 ml
?. [buffers]
Buffer W: 100 mM Tris-HCl (pH 8.0) 150 mM NaCl1 mM EDTABuffer E: 100 mM Tris-HCl (pH 8.0) 150 mM NaCl1 mM EDTA2.5 mM biotin Buffer E: 100 mM Tris-HCl (pH 8.0) 150 mM NaCl1 mM EDTA2.5 mM biotin
?. [Protein extraction]
The cells are sonificated (VS70 T rod, 25% 1 sec on 2 sec off for . On ice water). For small amounts use the MS72 rod. From this point be very sure to keep the cells on ice as much as possible.
?. [Protein extraction]
The cell extract is centrifuged for at 30 000 g.
?. The supernatant is collected and filtered first with 0.45 um filter and then with 0,22 um filter. If there are small sample volumes: add  buffer W.
3 ml
?. [Protein purification]
Fill centrifuge Column with 200-300 μL Strep- Tactinsepharose (so 400-600 μL 50% suspension). This is your CV.
?. [Protein purification]
Equilibrate with 3 Column bed Volume (CV) of Buffer W.
?. [Protein purification]
Load 10 CV of cell free extract on the column
?. [Protein purification]
Wash the resin 5 times by 1 CV of buffer WCollect this in Eppendorf tubes per 1 CV. Apply  of the first washing fraction and  of each subsequent fraction to an analytical SDS-PAGE.
2 µl
8 µl
?. [Protein purification]
Apply 0.5 CV of Buffer E on column 6 times to elute the protein of interest 8 μl samples of each fraction can be used for SDS-PAGE analysis. Most of the purified Strep-tag® II fusion protein usually elutes in the 2nd to 5th fraction.
?. [Protein purification]
Determine protein concentration flow through on nanodrop with the molecular weight and extinction coefficient
?. [Protein purification]
Run on an appropriate SDS-page gel
TITLE: RNA extraction from milk for HPAI surveillance
AUTHORS: Andrew Lail, Will Vuyk, Isla Emmen, David O'Connor

[DESCRIPTION]
This is an RNA extraction method that isolates HPAI RNA segments from commercially-available milk samples. The protocol involves processing with a MagMax RNA extraction kit on a Kingfisher instrument. Then, the samples are concentrated using a Zymo Clean and Concentrate kit. The total RNA yields should be suitable for performing qPCR. After trying other methods involving centrifugation, filtration, and trizol, we determined this method to be the easiest and most effective. 

[STEPS]
SECTION: RNA extraction
1. Use the MagMax Wastewater Ultra x96 kit1 (or comparable magnetic bead isolation kit2-5) on the ThermoFisher Kingfisher Apex (or comparable instrument). For each sample, load 400 uL of pasteurized milk or heavy whipping cream into the binding plate. 
SECTION: RNA extraction
2. Elute in 50 uL of elution solution or nuclease-free water.
SECTION: Optional Sample Concentration and Cleaning
6. Use a Zymo RNA Clean and Concentrator-5 kit7 to clean and concentrate the sample. Elute this into 30 uL final volume of nuclease-free water for downstream analysis.
SECTION: Optional Sample Concentration and Cleaning
4. Take 200 uL forward into the DNase treatment. Using ThermoFisher TurboDNase, combine 200 uL of pooled eluate, 22 uL 10X TurboDNase buffer, and 2 uL TurboDNase.
SECTION: Optional Sample Concentration and Cleaning
3. Pool the eluate from 6 extraction replicates of the same sample.
SECTION: Optional Sample Concentration and Cleaning
5. Incubate at 37 C for 30 minutes.
TITLE: Making LB agar plates
AUTHORS: Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from the LB within. When required the addition of an antibiotic to this gel allows for the selection of only those bacteria with resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance gene. </div><div class = "text-block"><span>See the link for more details: </span><a href="https://www.addgene.org/protocols/pouring-lb-agar-plates" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/pouring-lb-agar-plates/</span></a></div></div>

[STEPS]
?. Purchase LB Agar from the media kitchen and autoclave it to meltContent of LB Agar:
37 g pre-mixed powder consisting of:- 5 g peptone- 10 g peptone from casein- 10 g sodium chloride- 10 g g agar-agar1 L Sterile H2O
?. If the bacteria have a specific antibiotic resistance then add 1000x of that antibiotic to the melted LB agar before pouring, e.g. if we use ampicillin we add 100mg/ml to the LB in a way that the final concentration becomes 100ug/ml. So, for 200ml LB agar we add 200ul of 100mg/ul of stock ampicillin  ABC1  Antibiotic    Recommended Stock Concentration    Recommended Working Concentration  2  Ampicillin    100 mg/mL    100 µg/mL  3  Bleocin    5 mg/mL    5 µg/mL  4  Carbenicillin    100 mg/mL    100 µg/mL  5  Chloramphenicol    25 mg/mL(dissolve in EtOH)    25 µg/mL  6  Coumermycin    25 mg/mL(dissolve in DMSO)    25 µg/mL  7  Gentamycin    10 mg/mL    10 µg/mL  8  Kanamycin    50 mg/mL    50 µg/mL  9  Spectinomycin    50 mg/mL    50 µg/mL  10  Tetracycline    10 mg/mL    10 µg/mL
ABC1  Antibiotic    Recommended Stock Concentration    Recommended Working Concentration  2  Ampicillin    100 mg/mL    100 µg/mL  3  Bleocin    5 mg/mL    5 µg/mL  4  Carbenicillin    100 mg/mL    100 µg/mL  5  Chloramphenicol    25 mg/mL(dissolve in EtOH)    25 µg/mL  6  Coumermycin    25 mg/mL(dissolve in DMSO)    25 µg/mL  7  Gentamycin    10 mg/mL    10 µg/mL  8  Kanamycin    50 mg/mL    50 µg/mL  9  Spectinomycin    50 mg/mL    50 µg/mL  10  Tetracycline    10 mg/mL    10 µg/mL
?. Maintaining sterile techniques, pour the molten agar on large petri dishes (90mm) roughly about 30-45ml (don’t need to be exact on the volume) and leave to dry overnight
?. Store the plates at 4C to be used later, ideally it should be used within 2 weeks but can be used for longer if not contaminated or dehydrated but never more than 3months
TITLE: Chlamydomonas reinhardtii cell wall extraction with perchlorate
AUTHORS: Joao Vitor Molino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols describe the steps required for the perchlorate extraction of cell wall proteins from Chlamydomonas reinhardtii. </div><div class = "text-block">Protocol based on:</div></div>

[STEPS]
?. [Material]
Sodium PerchlorateCentrifuge tubesPipettes and tipsCentrifuge
?. [Perchlorate solution]
Sodium perchlorate has the following safety concerns.
?. [Cell preparation]
Grow cells until the beginning of the stationary phase. (Until obtained the highest amount of cells)Count the cells concentration, and harvest the target amount of cells by centrifugation  . (   to   per mL of   sodium perchlorate were tested)Remove the supernatant decantation or pipettingWash the cells with fresh TAP media, by ressuspending them in fresh media, followed by centrifugation  Remove the supernatant decantation or pipettingCount the cells concentration, and harvest the target amount of cells by centrifugation . (  to  per mL of  sodium perchlorate were tested)Remove the supernatant decantation or pipettingWash the cells with fresh TAP media, by ressuspending them in fresh media, followed by centrifugation  Remove the supernatant decantation or pipettingWash the cells with fresh TAP media, by ressuspending them in fresh media, followed by centrifugation
Centrifuge: 4000 1324, 10 min, 25 10
Centrifuge: 4000 1324, 10 min, 25 10
?. [Cell wall extraction]
Add the desired amount of sodium perchlorate solution. (ex. From a  culture, harvest by centrifugation   of culture, and after washing, add 150 uL of sodium perchlorate solution)Mix by vortexing or pipettingCentrifuge at max speed (ex. For smal volumes   . For larger volumes lower speed can be used for longer time    )Recover the supernatant containing the extracted proteins.Centrifuge at max speed (ex. For smal volumes  . For larger volumes lower speed can be used for longer time   )
1.5 mL
Centrifuge: 20000 1324, 1 min, 25 10
Centrifuge: 4000 1324, 10 min, 25 10