pHR-SFFV-dCas9-BFP-KRAB

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

CRISPRi screens
Growth screens
Several cell lines expressing dCas9-KRAB were obtained from previous publications:
HEK293T(22), HeLa(22), K562 (23), iPSCs (WTC-CRISPRi Gen IC)(33), and
U87(42). MCF7 and MDA-MB-231 were generated for this study by infecting lentivirus
expressing dCas9-KRAB-BFP (Addgene #46911; (22)) and sorting for single cell clones
stably expressing high BFP. Replicates for these cell lines were performed in different
clones. All cell lines except iPSCs were infected in duplicate with sgRNA the sublibraries
described above or the PVT1 tiling library, packaged with TransIT-LT1 (Mirus, Madison, WI) transfection in HEK293T cells (not expressing dCas9-KRAB), at an initial infection
rate of 30-50% (300-500x coverage of the library). Cells were cultured for two days following infection, treated for two days with 0.75-1.00 µg/mL puromycin, allowed to recover for one day, and then cultured at a minimum coverage of 1000x for 12 days (K562, HEK293T, U87, HeLa) or 20 days (MCF7, MDA-MB-231) starting from this
“T0.” K562 cells were passaged daily, while adherent cells were split on alternate days. iPSCs were infected at ~15% infection (with double the starting cell population to yield 300x coverage), grown for 3 days, selected with 1.5 µg/mL puromycin for 9 days, and allowed to recover for 3 days. iPSCs were then divided into two independent replicates and treated daily with 2µM doxycycline starting from this T0 and for the following 18
days (with primary endpoint at 12 days used here unless otherwise specified). Cells with a minimum of 1000x library coverage were harvested the day following puromycin recovery (T0) and at the endpoint, and processed for sequencing on Illumina HiSeq 2500
or 4000 as previously described(23, 35).

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Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Repression lncRNA PVT1 using pHR-SFFV-dCas9-BFP-KRAB from Addgene.

Manufacturer protocol

Download the product protocol from Addgene for pHR-SFFV-dCas9-BFP-KRAB below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Human - Repression lncRNA PVT1 using pHR-SFFV-dCas9-BFP-KRAB from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.