TITLE: HTAPP_Dissociation of human primary colorectal cancer resection specimens into single cell suspension for single cell RNA-seq
AUTHORS: Karin Pelka, Jonathan H Chen, Matan Hofree, Tabea Moll, Max Goder-Reiser, SusannahPhillips, Anise Applebaum, Danielle Dionne, Will Ge, Julia Waldman, Toni Delorey, Mike Cuoco, Moshe Biton, Moshe Sade-Feldman, Judit Jané-Valbuena, Ana Anderson, Vijay Kuchroo, Andrew Aguirre, Orit Rozenblatt-Rosen, Tatyana Sharova, Genevieve Boland, Aviv Regev, Nir Hacohen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the dissociation of primary human colorectal cancer (CRC) resection specimens into single cell suspensions for single cell RNA-seq applications. It was successfully used for droplet-based single cell RNA-seq of more than 60 patient specimens using the 10x Genomics platform, including 21 HTAPP CRC cohort samples. It can also be used upstream of other single cell RNA-seq approaches such as plate-based SS2 single cell RNA-seq protocols.</div></div>

[STEPS]
?. [Sample Description and Allocation]
Report sample information.
Sample ID:Time specimen out:Time start tissue triage:Other assays tissue is allocated to:
?. [Sample Description and Allocation]
Sample allocation: Transfer resection specimen into a Petri dish kept on ice. Take a picture of the specimen alongside a ruler. Remove fat, necrotic and fibrous areas. Wash off residual blood and stool using cold RPMI as needed. Dissect tissue using a scalpel in order to allocate tissue to assays as required. Weigh specimen allocated for scRNAseq using a precision scale. Work quickly. In order to weigh the tissue, carefully tap tissue onto a kimwipe to remove excess liquid and transfer onto a petri dish sitting in a precision scale. Move tissue back into petri dish on ice as quickly as possible and proceed to the next step immediately. If delays occur, make sure to cover the specimen with RPMI.
[wet ice]
Picture of sample:Description of sample appearance:Weight of specimen allocated for scRNAseq:
?. [Tissue Dissociation]
Mechanical dissociation: Mince tissue allocated for scRNASeq into small pieces (~1 mm^3) using a scalpel. Work quickly and keep the specimen in the petri dish on ice during this procedure. Mechanical dissociaton is critical to ensure efficient enzymatic digestion.
[wet ice]
?. [Tissue Dissociation]
Enzymatic digestion: Transfer dissociated tissue into 1 ml eppendorf tubes, each containing 1 ml of enzymatic digestion mix (Miltenyi, human Tumor Dissociation kit) containing:  AB1Enzyme H100 ul2Enzyme R50 ul3Enzyme A12.5 ul4RPMI 16402.35 mlUse 1 ml of digestion mix per 50 mg of tissue. Invert tubes to resuspend the tissue. Transfer eppendorf tubes to a rotation shaker set to 37˚C and 550 rpm and incubate shaking for 20 min.
AB1Enzyme H100 ul2Enzyme R50 ul3Enzyme A12.5 ul4RPMI 16402.35 ml
[rotation shaker, 550 rpm]
?. [Tissue Dissociation]
70 um filter step: Transfer digestion mix including any remaining tissue pieces onto a 70um cell strainer sitting on a 50 ml falcon tube on ice. Cut off the end of pipette tip and use low retention tips in order to prevent the tissue from sticking to the pipette tip. Flush the eppendorf tube once with RPMI containing 2% human serum. Use plunger of a 1 ml syringe in order to carefully squeeze the tissue pieces through the filter. Flush with RPMI containing 2% human serum as needed until the suspension has passed through the filter.
[wet ice]
?. [Tissue Dissociation]
Centrifugation: Centrifuge cell suspension at 500 g for 7 min in 4˚C pre-cooled centrifuge. Note: If the cells fail to pellet after this spin, repeat this step.
4 °C
Centrifuge: 500 34
?. [Red Blood Cell removal]
Carefully transfer supernatant into a 50 ml falcon labeled 'Supernatant' kept on ice. Try to remove as much liquid as possible without disturbing the pellet (usually keeping about 100-500 ul on the pellet). The supernatant will be discarded unless the pellet got disturbed in which case step 6 needs to be repeated with the supernatant tube.
4 °C
?. [Red Blood Cell removal]
Resuspend pellet in 4 ml of ACK lysis buffer and lyse red blood cells for 2 min on ice.
[wet ice]
?. [Red Blood Cell removal]
Stop ACK lysis by adding 26 ml of cold RPMI containing 2% human serum.
[wet ice]
?. [Red Blood Cell removal]
Centrifugation: Centrifuge cell suspension at 500 g for 7 min in 4˚C pre-cooled centrifuge. Note: If the cells fail to pellet after this spin, repeat this step.
4 °C
Centrifuge: 500 34
?. [Final cleanup]
Carefully transfer supernantant into a 50 ml falcon labeled 'Supernatant' kept on ice. Try to remove as much liquid as possible without disturbing the pellet (usually keeping about 100-500 ul on the pellet). The supernatant will be discarded unless the pellet got disturbed in which case step 10 needs to be repeated with the supernatant tube.
4 °C
?. [Final cleanup]
Resuspend cell pellet in cold 10x loading buffer (PBS containing 0.04% m/v BSA). 10x loading buffer should be freshly prepared on the day of the experiment. However, sterile filtered 4% m/v BSA in PBS stock solutions (2 g BSA in 50 ml PBS final volume) can be stored at 4˚C and diluted 1:100 at the day of the experiment. Adjust the volume to the size of the cell pellet, start with 500 ul and increase the volume if the cell suspension is too dense to filter. Use low retention tips.
[wet ice]
?. [Final cleanup]
Filter the cell suspension through a blue cap FACS filter top. If cell suspension is not passing through the filter, dilute the cell suspension and collect the cell suspension from the bottom of the filter using a pipette. Transfer the filtered cell suspension into an 1.5 ml eppendorf tube.
[wet ice]
?. [Final cleanup]
Centrifugation: Centrifuge cell suspension at 500 g for 2 min in 4˚C pre-cooled centrifuge. Use a swing out rotor in order to pellet cells at the bottom of the eppendorf tube.
4 °C
Centrifuge: 500 34
?. [Final cleanup]
Carefully transfer supernantant into a 1.5 ml eppendorf tube labeled 'Supernatant' kept on ice. Try to remove as much liquid as possible without disturbing the pellet (usually keeping about 10-20 ul on the pellet). The supernatant will be discarded unless the pellet got disturbed in which case step 14 needs to be repeated with the supernatant tube.
4 °C
?. [Final cleanup]
Resuspend the pellet in cold 10x loading buffer (PBS containing 0.04% m/w BSA). Judge by the size of the pellet how much volume to use, typically around 50-500 ul.
[wet ice]
?. [Quality control]
Mix 1 ul of single cell suspension with 9 ul of trypan blue and count live and dead cells using a hemocytometer. Report cell number and viability. Note: If cell viability is low or lots of debris is visible, go back to step 13 for an additional filter step. Viability should not be below 60%.
Cell number:Cell viability:Other observations (e.g. debris, cell clumps):
?. [10x loading]
Adjust cell concentration to 1E6 cells/ml (counting both live and dead cells) by diluting cell suspension in cold 10x loading buffer (PBS containing 0.04% BSA) and immediately proceed to 10x loading.Note: If more cells than needed are available, only a part of the cell suspension can be diluted for 10x loading and the remaining cell suspension can be used for other purposes.
?. [10x loading]
Report the time at which the sample is loaded onto the 10x machine, the person loading, and the concentration and number of cells loaded.
Time of Loading on 10x machine:Person Loading:10x kit Used:Numer of Cells Loaded per Channel:Number of Channels loaded:
?. [Optional: Post loading QC]
Note: For additional quality assessment, any left over cell suspension after 10x loading can be used for flow analysis. The flow staining panel should be adjusted based on the focus of the study and available cytometer. The staining procedure and panel described below can be used to assess the general cellular composition and is compatible with SONY SH800 cell sorters if sorting of specific cell populations is desired.
?. [Optional: Post loading QC]
Add 1 ml of cold PBS to remaining cell suspension to wash out any remaining BSA and centrifuge cell suspension at 500 g for 2 min in 4˚C pre-cooled centrifuge. Use a swing out rotor in order to pellet cells at the bottom of the eppendorf tube.
4 °C
Centrifuge: 500 34
?. [Optional: Post loading QC]
Live/Dead staining: Carefully remove as much liquid as possible without disturbing the pellet (usually keeping about 10-20 ul on the pellet) and resuspend the cell pellet in Zombie Violet Fixable Viability dye (Biolegend 423113, stock solution prepared in DMSO as recommended by the manufactorer) diluted 1:100 in cold PBS and incubate
4 °C
?. [Optional: Post loading QC]
Add 1 ml of cold PBS containing 0.5% BSA and 2mM EDTA to cell suspension and centrifuge cell suspension at 500 g for 2 min in 4˚C pre-cooled centrifuge. Use a swing out rotor in order to pellet cells at the bottom of the eppendorf tube.
4 °C
Centrifuge: 500 34
?. [Optional: Post loading QC]
Antibody staining: Carefully remove as much liquid as possible without disturbing the pellet (usually keeping about 10-20 ul on the pellet), resuspend the cell pellet in 100 ul of the following antibody master mix  and incubate   AB1Human TruStain FcX™ Fc Receptor Blocking Solution, Biolegend 4223015 ul2APC anti-human CD326 (EPCAM), Biolegend 3242071 ul3PE-Cy7 anti-human CD235a, Biolegend 3491121 ul4AF700 anti-human  CD45, Biolegend 3040231 ul5FITC anti-human CD2, Biolegend 3002031 ul6FITC anti-human CD3, Biolegend 3028051 ul7FITC anti-human CD19, Biolegend 3022061 ul8FITC anti-human CD20, Biolegend 3023041 ul9FITC anti-human CD56 Biolegend 3183041 ul10PE anti-human CD66b Antibody, Biolegend 3051051 ul11PBS containing 0.5% BSA and 2 mM EDTA86 ul
4 °C
AB1Human TruStain FcX™ Fc Receptor Blocking Solution, Biolegend 4223015 ul2APC anti-human CD326 (EPCAM), Biolegend 3242071 ul3PE-Cy7 anti-human CD235a, Biolegend 3491121 ul4AF700 anti-human  CD45, Biolegend 3040231 ul5FITC anti-human CD2, Biolegend 3002031 ul6FITC anti-human CD3, Biolegend 3028051 ul7FITC anti-human CD19, Biolegend 3022061 ul8FITC anti-human CD20, Biolegend 3023041 ul9FITC anti-human CD56 Biolegend 3183041 ul10PE anti-human CD66b Antibody, Biolegend 3051051 ul11PBS containing 0.5% BSA and 2 mM EDTA86 ul
?. [Optional: Post loading QC]
Add 1 ml of cold PBS containing 0.5% BSA and 2mM EDTA to cell suspension and centrifuge cell suspension at 500 g for 2 min in 4˚C pre-cooled centrifuge. Use a swing out rotor in order to pellet cells at the bottom of the eppendorf tube.
4 °C
Centrifuge: 500 34
?. [Optional: Post loading QC]
Measure on flow cytometer.