Cell Fixation
HiTIF Protocols
Access HiTIF protocols, downloadable SOPs, and Optical Pooled Screening workflow documentation.
Cell Seeding
Seeding Cells in the imaging plates using the Multidrop Combi
Download FileFunctional Genomic Screens
RNAi/CRISPR-KO Screen Assay Development SOP
Download FilesiRNA/sgRNA Spotting
Optimization of reverse transfection conditions for siRNA or sgRNA oligos
Download FilesiRNA/sgRNA Transfection
Reverse siRNA/sgRNA Transfection Protocol for Users
Download FileOptical Pooled Screening SOPs
A complete set of standard operating procedures for HiTIF’s pooled HCI platform, covering cell-line preparation, lentiviral library generation, in situ sequencing library preparation, sequencing, and imaging.
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OPS SOP Master Index
Full SOP listing, estimated times, prerequisites, and workflow dependencies.
Download IndexOverview
OPS Workflow Walkthrough
Narrative overview of the end-to-end pooled screening workflow.
Download WalkthroughOPS Workflow
- Cell Line Preparation
- Lentivirus
- Cell Library Generation
- OPS Library Preparation
- Sequencing
- Imaging
- Reagents
SOP Directory
Cell Line Preparation 3 SOPs
SOP 001 — Generation of Inducible Cas9 Cell Lines
PiggyBAC-based generation of a stable, doxycycline-inducible Cas9 cell line.
SOP 002 — Isolating Monoclonal Cell Populations
Limiting-dilution cloning to isolate monoclonal populations.
SOP 006 — Cas9 Efficiency Measurement
Validation of iCas9 activity using an EGFP reporter.
Lentivirus 3 SOPs
SOP 003 — Lentivirus Production
High-titer lentivirus generation in HEK293T cells.
SOP 004 — Lentivirus Titration
Determination of functional viral titer.
SOP 005 — Lentivirus Transduction
Transduction of target cells with lentivirus.
Cell Library Generation 2 SOPs
SOP 007 — Generation of Cell Libraries
Lentiviral transduction and pooled cell-library creation.
SOP 008 — CRISPR Library Validation
sgRNA representation analysis of the generated library.
OPS Library Preparation 2 SOPs
SOP 009a — Conventional OPS Library Preparation
Cell fixation, reverse transcription, padlock probes, and rolling-circle amplification.
SOP 009b — T7 Library Preparation
Alternative library preparation using T7 polymerase.
Sequencing 1 SOP
SOP 010 — SBS Illumina Sequencing
In situ sequencing by synthesis.
Imaging 1 SOP
SOP 011 — Imaging Using Nikon Ti2
Phenotype and in situ sequencing image acquisition on the Nikon Ti2.
Reagents and Oligonucleotides
OPS Reagents
Master reagent lists and oligonucleotides.
OPS Cell Library
Cell-library worksheet resource.