Cloning Details

[gwaybio]

As discussed at the meeting today, we were curious about the cloning details. We can use this issue to elaborate on the cloning procedure. We are specifically interested in knowing more details about how resistance was identified and selected for.

cc @mekelley @ayberman

[ayberman]

Hi Greg,

The clones were selected using two different procedures for WT and Bortezomib resistant clones. Here's what we did:

WT:
Parental HCT116 cells were diluted to a concentration of 5cells/mL in conditioned media, and plated in a 96 well plate (100uL/well, for <1 cell per well in order to get some wells with exactly 1 cell).

The cells were grown for 2 weeks, until single colonies were observed. The media was replaced every ~3 days.
->any well with more than one colony was excluded, as these colonies may have grown from 2 different cells

The colonies were transferred to larger plates for expansion of the clonal cell line.

Bortezomib resistant:
Parental HCT-116 cells were grown to near confluence on a 20 cm dish and then treated with 12 nM bortezomib for 3 weeks. Media + bortezomib was replaced every 4-6 days. Over the course of the 3-week treatment, almost all parental cells died, leaving spatially separated, bortezomib-resistant, single cells, which grew into colonies. Colonies were isolated using clonal rings and expanded for use.

Let us know if you have any questions!

[gwaybio]

This is AMAZING! thank you 🚀