Generation of NPC1-knockout (KO) cell lines

IK Ilyas Khan
SL Sunan Li
LT Lihong Tao
CW Chong Wang
BY Bowei Ye
HL Huiyu Li
XL Xiaoyang Liu
IA Iqbal Ahmad
WS Wenqiang Su
GZ Gongxun Zhong
ZW Zhiyuan Wen
JW Jinliang Wang
RH Rong-Hong Hua
AM Ao Ma
JL Jie Liang
XW Xiao-Peng Wan
ZB Zhi-Gao Bu
YZ Yong-Hui Zheng
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Oligos encode small guide (sg) RNAs that target NPC1 genes in human cells, Vero-E6 cells, and CHO cells (listed in Supplementary Table 4) were cloned into LentiGuide-Puro vector after BsmBI digestion. Lentiviruses were produced from HEK293T cells by transfection of LentiGuide-Puro, pMD2.G, and psPAX2 at a ratio of 4:3:2 using PEI. After infecting cells for 48 h, cells were selected by treatment with puromycin at 10 µM for CHO cells, 5 µM for A549 cells, 6 µM for Caco2 cells, 10 µM for Vero-E6 cell, and 2 µM for Calu3 cells. Single clones of CHO, A549, Caco2 and Vero-E6 were sorted in 96-well plates using Beckman Coulter MoFlo XDP cell sorter. Calu3 was used directly after treatment by puromycin. NPC1-KO cells were identified by western blotting.

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