TITLE: Nuclei prep for single cell RNA/ATAC seq from intestinal surgical samples
AUTHORS: Ran Zhou, Oni Basu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Isolating high-quality nuclei from intestinal surgical tissues is critical for single cell RNA/ATAC-seq. This protocol provides details on nuclei preparation for such application, with an overall working time of less than an hour, if FACS sorting is not incorporated.</div></div>

[STEPS]
?. [Pre-processing of surgical samples]
Rinse surgical samples in ice-cold PBS for three times.
?. [Pre-processing of surgical samples]
Place the surgical samples in a 100 mm petri dish with cold HBSS, and isolate mucosa layer by Iris scissors.
?. [Pre-processing of surgical samples]
Weigh mucosa layer and cut the tissue into small pieces (
?. [Pre-processing of surgical samples]
Place each small piece in a 1.7 ml Eppendorf tube on ice.
Flash freeze the unused tissus in liquid nitrogen and store those tissues in liquid nitrogen/at - 80 Celsius.
?. [Lyse surgical tissues for nuclei]
Mince the tissue (fresh or frozen) in 0.5 ml lysis buffer by Iris Scissors on ice for 5 minutes.
on ice
?. [Lyse surgical tissues for nuclei]
Transfer the minced tissue in lysis buffer to a 5 ml conical tube. Add an additional 3 ml lysis buffer to the tube.
?. [Lyse surgical tissues for nuclei]
Mix the tissue with lysis buffer by inverting the tube and incubate on ice for 10 minutes.
on ice
Agitate tissue in lysis buffer by inverting the tube every 2 minutes.
?. [Enrich nuclei from the lysate]
Wet a 100 micron cell strainer with 1 ml lysis buffer.
?. [Enrich nuclei from the lysate]
Filter the lysate through the strainer on ice and wash the strainer with 3 ml NSB. Keep the flow through as this is where your nuclei are.
on ice
If FACS sorting is incorporated after nuclei enrichment, NSB in this experiment is supplemented with Hoechst 33342 10 ug/ml and WGA-Texas Red 1 ug/ml.
?. [Enrich nuclei from the lysate]
Wet a 40 micron cell strainer with 1 ml lysis buffer.
?. [Enrich nuclei from the lysate]
Filter the flow-through on this strainer on ice and wash the strainer by 3 ml NSB. Keep the flow through as this is where your nuclei are.
on ice
?. [Enrich nuclei from the lysate]
Spin the final flow-through in a 15 ml conical tube, 500 g x 5 minutes at 4 Celsius.
Centrifuge: 500 34, 5 min, 4 10
?. [Enrich nuclei from the lysate]
For fresh tissues, suspend the pelleted nuclei in 1 ml NSB with gentle pipetting and continue with step 14.For frozen tissues, suspend the pelleted nuclei in 200 ul NSB with gentle pipetting and continue with step 18.
?. [Enrich nuclei from the lysate]
Add RBC lysis buffer 10 ml to the fresh tissue nuclei suspension.
?. [Enrich nuclei from the lysate]
Invert the tube three times to mix and incubate at room temperature for 2 minutes.
The incubation may be extended (up to 10 minutes) to fully lyse RBCs.
?. [Enrich nuclei from the lysate]
Spin the nuclei suspension in a 15 ml conical tube, at 600 g x 5 minutes at 4 Celsius.
Centrifuge: 500 34, 5 min, 4 10
?. [Enrich nuclei from the lysate]
Suspend pelleted nuclei in 200 ul NSB or other nuclei suspension buffer as recommended by the single cell platform. Use gentle pipetting to resuspend as the nuclei are very fragile.
?. [Prepare nuclei for down stream applications]
Stain the nuclei with Hoechst 33342 10 ug/ml and WGA-Texas Red 1 ug/ml. Count the nuclei.
Skip this staining step for nuclei prepared for sorting.
?. [Prepare nuclei for down stream applications]
Adjust the nuclei concentration to the desired concentration using nuclei suspension buffer.
?. [Prepare nuclei for down stream applications]
Proceed with downstream applications.
After FACS sorting, nuclei are spun down at 700 g x 5 minutes at 4 celsius and stained by Hoechst 33324 10 ug/ml. Count the nuclei again and adjust the nuclei concentration accordingly.