Abstract
An erratum was issued for: Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing. The Protocol section was updated.
Step 3.1 was updated from:
Place the lumbar spinal cord in a pre-chilled Dounce homogenizer and add 500 mL pre-chilled detergent lysis buffer.
to:
Place the lumbar spinal cord in a pre-chilled Dounce homogenizer and add 500 μL pre-chilled detergent lysis buffer.
Step 3.6 was updated from:
Pass an additional 1 mL low sucrose buffer over the 40 mm strainer, bringing the final volume to 3 mL of the low sucrose buffer and 500 mL of the lysis buffer.
to:
Pass an additional 1 mL low sucrose buffer over the 40 mm strainer, bringing the final volume to 3 mL of the low sucrose buffer and 500 μL of the lysis buffer.
Step 5.5 was updated from:
Once the centrifugation is complete, immediately decant the supernatant in a flicking motion.
NOTE: A residual volume (less than 400 mL) of sucrose buffer can be discarded if desired to produce a lower volume and cleaner final sample, but this residual volume does contain nuclei and can be preserved to maximize nuclei yield
to:
Once the centrifugation is complete, immediately decant the supernatant in a flicking motion.
NOTE: A residual volume (less than 400 μL) of sucrose buffer can be discarded if desired to produce a lower volume and cleaner final sample, but this residual volume does contain nuclei and can be preserved to maximize nuclei yield
Step 5.6 was updated from:
Using 100 mL - 1 mL of resuspension solution, resuspend the nuclei remaining on the wall. Avoid the myelin ‘frown’ that remains with the detergent-based preparation.
to:
Using 100 μL - 1 mL of resuspension solution, resuspend the nuclei remaining on the wall. Avoid the myelin ‘frown’ that remains with the detergent-based preparation.
Steps 6.1.1 - 6.1.4 were updated from:
- Adjust nuclei to a final concentration of 225 nuclei per mL.
- Prepare barcoded beads at a concentration of 250 beads per mL.
- Prepare the lysis buffer with 0.7% sarkosyl.
- Adjust the flow rates to 35 mL per min for beads, 35 mL per min for nuclei, and 200 mL per min for oil.
to:
- Adjust nuclei to a final concentration of 225 nuclei per μL.
- Prepare barcoded beads at a concentration of 250 beads per μL.
- Prepare the lysis buffer with 0.7% sarkosyl.
- Adjust the flow rates to 35 μL per min for beads, 35 μL per min for nuclei, and 200 μL per min for oil.
Protocol
An erratum was issued for: Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing. The Protocol section was updated.
Step 3.1 was updated from:
Place the lumbar spinal cord in a pre-chilled Dounce homogenizer and add 500 mL pre-chilled detergent lysis buffer.
to:
Place the lumbar spinal cord in a pre-chilled Dounce homogenizer and add 500 μL pre-chilled detergent lysis buffer.
Step 3.6 was updated from:
Pass an additional 1 mL low sucrose buffer over the 40 mm strainer, bringing the final volume to 3 mL of the low sucrose buffer and 500 mL of the lysis buffer.
to:
Pass an additional 1 mL low sucrose buffer over the 40 mm strainer, bringing the final volume to 3 mL of the low sucrose buffer and 500 μL of the lysis buffer.
Step 5.5 was updated from:
Once the centrifugation is complete, immediately decant the supernatant in a flicking motion.
NOTE: A residual volume (less than 400 mL) of sucrose buffer can be discarded if desired to produce a lower volume and cleaner final sample, but this residual volume does contain nuclei and can be preserved to maximize nuclei yield
to:
Once the centrifugation is complete, immediately decant the supernatant in a flicking motion.
NOTE: A residual volume (less than 400 μL) of sucrose buffer can be discarded if desired to produce a lower volume and cleaner final sample, but this residual volume does contain nuclei and can be preserved to maximize nuclei yield
Step 5.6 was updated from:
Using 100 mL - 1 mL of resuspension solution, resuspend the nuclei remaining on the wall. Avoid the myelin ‘frown’ that remains with the detergent-based preparation.
to:
Using 100 μL - 1 mL of resuspension solution, resuspend the nuclei remaining on the wall. Avoid the myelin ‘frown’ that remains with the detergent-based preparation.
Steps 6.1.1 - 6.1.4 were updated from:
- Adjust nuclei to a final concentration of 225 nuclei per mL.
- Prepare barcoded beads at a concentration of 250 beads per mL.
- Prepare the lysis buffer with 0.7% sarkosyl.
- Adjust the flow rates to 35 mL per min for beads, 35 mL per min for nuclei, and 200 mL per min for oil.
to:
- Adjust nuclei to a final concentration of 225 nuclei per μL.
- Prepare barcoded beads at a concentration of 250 beads per μL.
- Prepare the lysis buffer with 0.7% sarkosyl.
- Adjust the flow rates to 35 μL per min for beads, 35 μL per min for nuclei, and 200 μL per min for oil.
Disclosures
No conflicts of interest declared.