TITLE: Dissociation of Jejunum cells for clumps sorting
AUTHORS: Rita Manco, Inna Averbukh, Ziv Porat, Keren Bahar Halpern, Ido Amit, Shalev Itzkovitz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Single-cell RNA sequencing combined with spatial information on landmark genes enables the reconstruction of spatially-resolved tissue cell atlases. However, such approaches are challenging for rare cell types since their mRNA contents are diluted in the spatial transcriptomics bulk measurements used for landmark gene detection. To overcome the limitations in reconstructing spatial expression profiles of rare cells, we present ClumpSeq, an approach for sequencing small clumps of attached tissue cells. Sequencing clumps increase the capture rate of rare cell types without the need for antibody enrichment and utilizes the spatial information of the major tissue cell type. We use this approach to reconstruct spatial maps of all intestinal secretory epithelial cell types along the crypt-villus axis, revealing zonated immune-modulatory programs and heterogeneous migration patterns. ClumpSeq can be applied for reconstructing spatial atlases of rare cell types in other tissues and tumors.</div></div>

[STEPS]
?. [Dissociation of Jejunum cells for clumps sorting]
Prepare buffers:Buffer1 (25ml): DPBS (-/-) 24.5 mlEDTA 0.5M (10mM) 500 µlBuffer2 (50ml): For Hoechst DMEM high glucose 44.5 ml10% FBS 5 mlHepes 1M (10mM) 500 µl
DPBS (-/-) 24.5 mlEDTA 0.5M (10mM) 500 µl
DMEM high glucose 44.5 ml10% FBS 5 mlHepes 1M (10mM) 500 µl
?. [Dissociation of Jejunum cells for clumps sorting]
Set a centrifuge to .
4 °C
?. [Dissociation of Jejunum cells for clumps sorting]
Warm at  of buffer1 and  of buffer2.
37 °C
15 mL
5 mL
?. [Dissociation of Jejunum cells for clumps sorting]
Prepare a tube with cold DPBS and put it  .
on ice
?. [Dissociation of Jejunum cells for clumps sorting]
Thaw the FBS.
IMPORTANT: The whole procedure needs to be done on ice!!!.
?. [Dissociation of Jejunum cells for clumps sorting]
Collect the tissue: proximal Jejunum - take  of tissue, starting  distal of the stomach.
8
8
?. [Dissociation of Jejunum cells for clumps sorting]
Wash the tissue: use a syringe filled with DPBS to flush the inside.
?. [Dissociation of Jejunum cells for clumps sorting]
Lateralize the tissue to flat it.
?. [Dissociation of Jejunum cells for clumps sorting]
Remove adjacent fat.
?. [Dissociation of Jejunum cells for clumps sorting]
Incubate the tissue for  in buffer1 .
on ice
?. [Dissociation of Jejunum cells for clumps sorting]
Move the tissue in the pre-warmed buffer1 and cut it into small pieces (~).
1
?. [Dissociation of Jejunum cells for clumps sorting]
Add  of DNase I in the tube.
1
?. [Dissociation of Jejunum cells for clumps sorting]
Incubate for  at  - shake every minute.
37 °C
?. [Dissociation of Jejunum cells for clumps sorting]
Filter the solution through a  mesh.
100
?. [Dissociation of Jejunum cells for clumps sorting]
Spin down in the cold centrifuge () for  at .
4 °C
300 g
?. [Dissociation of Jejunum cells for clumps sorting]
Discard the supernatant.
?. [Dissociation of Jejunum cells for clumps sorting]
Resuspend the pellet in  cold DPBS +  FBS and use the pipette to break the pellet gently.
8 mL
2 mL
?. [Dissociation of Jejunum cells for clumps sorting]
Filter the solution through  mesh.
100
?. [Dissociation of Jejunum cells for clumps sorting]
Spin down in the cold centrifuge () for  at .
4 °C
300 g
?. [Dissociation of Jejunum cells for clumps sorting]
Discard the supernatant.
?. [Dissociation of Jejunum cells for clumps sorting]
Resuspend the pellet in  of pre-warmed buffer2 with Hoechst (1:500) + Reserpine (1:4000) for  a  (keep it in the dark).
5 mL
37 Room temperature
?. [Dissociation of Jejunum cells for clumps sorting]
Spin down in a cold centrifuge () for  at .
4 °C
300 g
?. [Dissociation of Jejunum cells for clumps sorting]
Discard supernatant.
?. [Dissociation of Jejunum cells for clumps sorting]
Resuspend in DPBS and add 1:500 Zombie for  (Zombie works only in DPBS!!!).
37 Room temperature
?. [Dissociation of Jejunum cells for clumps sorting]
Wash 2x ().
300 g
4 °C
?. [Dissociation of Jejunum cells for clumps sorting]
Discard the supernatant.
?. [Dissociation of Jejunum cells for clumps sorting]
Resuspend the pellet in FACS buffer (accordingly to the pellet).
The sample is ready for the FACS sorting!!!