TITLE: Processing fallopian tube tissue for single cell sequencing analysis
AUTHORS: Ramlogan Sowamber, Melissa Castillo, iru.paudel , Sophia George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>This protocol explains the steps used to isolate cells for single cell sequencing from freshly processed fallopian tube tissue or cells that have been frozen. </span><span style = "font-weight:bold;">Step 1</span><span> outlines steps for digesting fallopian tube tissue. This is an overnight process using pronase which must not exceed 72 hours.  </span><span style = "font-weight:bold;">Step 2</span><span> outlines steps to process cells from </span><span style = "font-weight:bold;">Step 1</span><span> for single cell sequencing. </span><span style = "font-weight:bold;">Step 3</span><span> parallels </span><span style = "font-weight:bold;">Step 2</span><span>, but details processing cryopreserved cells prepared in </span><span style = "font-weight:bold;">Step 1.13</span><span>. </span><span style = "font-weight:bold;">Step 4</span><span> details steps for assessing viability and cell numbers, which will subsequently be used for single cell sequencing. In </span><span style = "font-weight:bold;">Step 5</span><span>, remaining cells from single cell sequencing can be plated and grown for downstream experiments or cryopreservation.</span></div></div></div>

[STEPS]
?. [Isolating cells from dissociated fallopian tube tissue]
In this step, cells isolated from fallopian tube tissues in Step 1.13 are in the form of a pellet within a 15 mL conical tube and are ready to proceed onto the following steps.
?. [Isolating cells from dissociated fallopian tube tissue]
Gently re-suspend pellet with 500 uL of cell culture media to dissociate pellet
?. [Isolating cells from dissociated fallopian tube tissue]
Pre-wet a 5 mL polystyrene round-bottom tube with cell strainer cap with 50 uL of cell culture media
?. [Isolating cells from dissociated fallopian tube tissue]
Allow liquid to flow through by gravity (if liquid does not flow through, either pipette liquid up and down to allow liquid to flow through or seal cap tube with parafilm and centrifuge tube at 300 x g for 1 min)
?. [Isolating cells from dissociated fallopian tube tissue]
Add 500 uL of resuspended cells from Step 2.1 to pre-wetted cell strainer cap and allow cells to flow through by gravity into 5 mL tube
?. [Isolating cells from dissociated fallopian tube tissue]
Place parafilm on cap to prevent contamination when taken out of the biological safety cabinet
?. [Isolating cells from cryopreserved fallopian tube cells]
In this step, cells previously frozen and kept at -80°C or liquid nitrogen from Step 1.13 can be thawed and processed
?. [Isolating cells from cryopreserved fallopian tube cells]
Thaw cryo-vial rapidly into 5 mL pre-warmed cell culture USG media.
?. [Isolating cells from cryopreserved fallopian tube cells]
Centrifuge cells at 300 x g for 5 minutes
?. [Isolating cells from cryopreserved fallopian tube cells]
Aspirate supernatant
?. [Isolating cells from cryopreserved fallopian tube cells]
Re-suspend cells gently in 500 uL of cell culture media
?. [Isolating cells from cryopreserved fallopian tube cells]
Pre-wet a 5 mL polystyrene round-bottom tube with cell strainer cap with 50uL of cell culture media
?. [Isolating cells from cryopreserved fallopian tube cells]
Allow liquid to flow through by gravity (if liquid does not flow through, either pipette liquid up and down to allow liquid to flow through or seal cap tube with parafilm and centrifuge tube at 300 x g for 1 min)
?. [Isolating cells from cryopreserved fallopian tube cells]
Add 500 uL of resuspended cells from Step 3.4 to pre-wetted cell strainer cap and allow cells to flow through by gravity into 5 mL tube
?. [Isolating cells from cryopreserved fallopian tube cells]
Place parafilm on cap to prevent contamination when taken out of the biological safety cabinet
?. [Isolating cells from cryopreserved fallopian tube cells]
Place tubes with cells in an incubator (37°C and 5% CO2) for 15 minutes
?. [Isolating cells from cryopreserved fallopian tube cells]
Proceed to Step 4.
?. [Isolating cells from dissociated fallopian tube tissue]
Proceed to Step 4
?. [Analyzing cell viability and quantifying cell numbers]
This step is used for assessing cell viability and counting the number of cells prior to sequencing
?. [Analyzing cell viability and quantifying cell numbers]
Count cells using the Nexcelom Cellometer K2 or equivalent cell counter
?. [Analyzing cell viability and quantifying cell numbers]
Assess viability and cell numbers before proceeding to sequencing
?. [Plating cells for growth]
At this step, the required volume of sample has been used for sequencing. If cell sample remains after single cell sequencing, proceed with the following steps.
?. [Plating cells for growth]
Prepare a 24 well Primaria plate for remaining cells by coating with 250 uL of collagen
?. [Plating cells for growth]
Aspirate excess collagen
?. [Plating cells for growth]
Add 250 uL of cell culture media to each well
?. [Plating cells for growth]
Remove cells from incubator (placed in incubator from Step 4.3) and very gently re-suspend liquid containing cells
?. [Plating cells for growth]
Add cells to a labelled well of the 24 well collagen coated Primaria plate using a pipette
?. [Plating cells for growth]
Gently move plate back and forth to evenly distribute cells
?. [Plating cells for growth]
Place cells in incubator (37°C and 5% CO2) and allow to grow.
?. [Tissue Dissociation Protocol]
Step 1.1 to 1.13 details steps for digesting human fallopian tube tissue using pronase.
?. [Tissue Dissociation Protocol]
Collect the fallopian tube tissue within two hours of removal. Samples are transported from the operating room/hospital in DMEM medium with 10% FBS (transport media) on ice
?. [Tissue Dissociation Protocol]
In a biological safety cabinet with appropriate PPE, aspirate the transport medium leaving behind 5 mL of media.
?. [Tissue Dissociation Protocol]
Place tissue into cell culture dish and use blade to finely mince tissue. DO NOT USE MORTAR AND PESTLE. Meanwhile label 50 mL conical tube with identifying information.
?. [Tissue Dissociation Protocol]
Using a 25 mL pipette, transfer minced tissue to 50 mL conical tube.
?. [Tissue Dissociation Protocol]
Add 15 mL of pronase solution to 50 mL tube. Add 150 uL of DNase to 15 mL solution.
?. [Tissue Dissociation Protocol]
Place 50 mL tube containing minced tissue on an orbital shaker at 4 °C and allow to digest for a minimum 24 hours and maximum 72 hours.
?. [Tissue Dissociation Protocol]
Proceed to step 1.8 after 24 - 72 hours. DO NOT LEAVE TISSUE IN PRONASE FOR MORE THAN 72 hours.
?. [Post tissue digestion processing ]
Remove 50 mL tube from 4 °C
?. [Analyzing cell viability and quantifying cell numbers]
Place remaining cells in 37°C incubator until ready to proceed to Step 5. Do not leave cells in incubator for more than 10 minutes.
?. [Isolating cells from dissociated fallopian tube tissue]
Place tubes with cells in an incubator (37°C and 5% CO2) for 15 minutes
?. [Post tissue digestion processing ]
If you would like to proceed with storing the live cells, carefully remove the supernatant and re-suspend the cell pellet in 1 mL of freezing media and cryopreserve cells in a -80°C freezer (transfer to liquid nitrogen within 48 hours). The number of cryovials used for cell cryopreservation depends on the size of the tissue. Otherwise gently aspirate supernatant and proceed to Step 2.
?. [Post tissue digestion processing ]
Add approximately 20 mL of RPMI solution to tube which should amount to no more than 40 mL of total media.
?. [Post tissue digestion processing ]
Gently shake 50 mL tube back and forth to mix media
?. [Post tissue digestion processing ]
Place 50 mL tube into a rack and allow the tissue to settle to the bottom of the tube.
?. [Post tissue digestion processing ]
Spin both tubes in centrifuge at 1500 rpm for 15 minutes at 4°C