TITLE: Perturb-seq characterizing regulators of T cell function
AUTHORS: Andrea R Daniel

[DESCRIPTION]
This protocol describes methods for a Perturb-seq assay characterizing transcriptional regulators of T cell function. 

[STEPS]
SECTION: gRNA library cloning
1. Oligonucleotide pools containing 40-gRNA sequences and constant regions for polymerase chain reaction (PCR) amplification were synthesized by Twist Bioscience. 
SECTION: gRNA library cloning
2. gRNA amplicons were gel extracted, PCR purified and input into 20 μl Gibson reactions (5:1 molar ratio of insert to backbone) with 200 ng of Esp3I digested and 1 × solid-phase reversible immobilization (SPRI)-selected (Beckman Coulter) plasmid backbone. 
SECTION: gRNA library cloning
3. Gibson reactions were purified using ethanol precipitation and transformed into Lucigen’s Endura ElectroCompetent Cells. 
SECTION: gRNA library cloning
4. Transformed cells were cultured overnight and plasmids were isolated using Qiagen Midi Kits.
SECTION: Transfections for high-titer lentiviral production
5. Plate 1.2 x 106 or 7 x 106 HEK293T cells in a 6 well plate or 10 cm dish in the afternoon with 2 mL or 12 mL of complete opti-MEM (Opti-MEM‱ I Reduced Serum Medium supplemented with 1x  Glutamax, 5% FBS, 1 mM Sodium Pyruvate, and 1x MEM Non-Essential Amino Acids).
SECTION: gRNA library cloning
2.1. Addgene: pLV hU6-gRNA hUbC-dSaCas9-KRAB-T2A-Thy1.1 (Addgene 194278) and pLV hU6-gRNA hUbC-VP64-dSaCas9-VP64-T2A-Thy1.1 (Addgene 194279).
SECTION: Transfections for high-titer lentiviral production
6. The next morning, transfect HEK293T cells with 0.5 μg pMD2.G, 1.5 μg psPAX2, and 0.5 μg transgene for 6 well plates or 3.25 μg pMD2.G, 9.75 μg psPAX2, and 4.3 μg transgene for 10 cm dishes using Lipofectamine 3000.
SECTION: Transfections for high-titer lentiviral production
7. Exchanged media 6 hours after transfection and collect and pool lentiviral supernatant at 24 hours and 48 hours after transfection.
SECTION: Transduction of primary human T cells
8. Centrifuged lentiviral supernatant at 600g for 10 min to remove cellular debris. 
SECTION: Transduction of primary human T cells
9. Concentrate lentivirus to 50–100× the initial concentration using Lenti-X Concentrator (Takara Bio). 
SECTION: Transduction of primary human T cells
10. Transduce T cells at 5–10% v/v of concentrated lentivirus at 24 h post-activation. For dual transduction experiments, T cells were serially transduced at 24 h and 48 h.
SECTION: scRNAseq
11. CD8+CCR7+ T cells from three donors were transduced with CRISPRi and CRISPRa mini-TF gRNA libraries.
SECTION: scRNAseq
12. T cells were expanded for 10 days and then stained and sorted for Thy1.1+ cells (a marker to identify transduced cells). 
SECTION: scRNAseq
16. Sorted cells were loaded into the Chromium X for a targeted recovery of 2 × 104 cells per donor and treatment according to the Single Cell 5′-High-Throughput Reagent Kit v2 protocol (10x Genomics). 
SECTION: scRNAseq
13. An SH800 FACS Cell Sorter (Sony Biotechnology) was used for cell sorting and analysis.
SECTION: scRNAseq
12.1. 
Antibody: Thy1.1-PE, clone OX-7, Mouse IgG1, κ, 1:300, Flow cytometry,	StemCell Technologies,	60024PE	
SECTION: scRNAseq
14. For antibody staining of Thy1.1 cells were collected, spun down at 300g for 5 min, resuspended in flow buffer (1× phosphate-buffered saline (PBS), 2 mM ethylenediaminetetraacetic acid and 0.5% bovine serum albumin) with the appropriate antibody dilutions and incubated for 30 min at 4 °C on a rocker. 
SECTION: scRNAseq
15. Cells were then washed with flow buffer, spun down at 300g for 5 min and resuspended in flow buffer for cell sorting.
SECTION: scRNAseq
17. SaCas9 gRNA sequences were captured by spiking in 2 μM of a custom primer into the reverse transcription master mix, as previously done for SpCas9 gRNA capture36. The custom primer was designed to bind to the constant region of SaCas9’s gRNA scaffold.
SECTION: scRNAseq
18. 5′-Gene Expression (GEX) and gRNA libraries were separated using double-sided SPRI bead selection in the initial cDNA clean-up step. 5′-GEX libraries were constructed according to manufacturer’s protocol.
SECTION: scRNAseq
19. gRNA libraries were constructed using two sequential PCRs (PCR 1: 10 cycles, PCR 2: 25 cycles). The PCR 1 product was purified using double-sided SPRI bead selection at 0.6 × and 2 ×. Twenty percent of the purified PCR 1 product was input into PCR 2. The PCR2 product was purified using double-sided SPRI bead selection at 0.6 × and 1 ×. 
SECTION: scRNAseq
20. All libraries were run on a High Sensitivity D1000 tape to measure the average amplicon size and quantified using Qubit’s dsDNA High Sensitivity assay. 
SECTION: scRNAseq
21.  Libraries were individually diluted to 20 nM, pooled together at desired ratios and sequenced on an Illumina NovaSeq S4 Full Flow Cell (200 cycles) with the following read allocation: Read 1, 26; i7 index, 10; Read 2, 90. 
SECTION: scRNAseq
22. All oligos are available in Supplementary Table 5 of McCutcheon et al. Nature Genetics, 2023. https://doi.org/10.1038/s41588-023-01554-0
SECTION: Processing and analyzing scRNA-seq
23. CellRanger v6.0.1 was used to process, demultiplex and generate UMI counts for each transcript and gRNA per cell barcode. 
SECTION: Processing and analyzing scRNA-seq
24. UMI counts tables were extracted and used for subsequent analyses in R using the Seurat69 v4.1.0 package.
SECTION: Processing and analyzing scRNA-seq
25. Low-quality cells with <200 detected genes, >20% mitochondrial reads or <5% ribosomal reads were discarded.
SECTION: Processing and analyzing scRNA-seq
26. DoubletFinder70 was used to identify and remove predicted doublets. All remaining high-quality cells across donors for each treatment (CRISPRi or CRISPRa) were aggregated for further analyses. 
SECTION: Processing and analyzing scRNA-seq
27. gRNAs were assigned to cells if they met the threshold (gRNA UMI >4). Cells were then grouped on the basis of gRNA identity. 
SECTION: Processing and analyzing scRNA-seq
28. For differential gene expression analysis, we compared the transcriptomic profiles of cells sharing a gRNA to cells with only NT gRNAs using Seurat’s FindMarkers function to test for DEGs with the hurdle model implemented in model-based analysis of single-cell transcriptomics (MAST).
SECTION: Processing and analyzing scRNA-seq
29. All significant gRNA–gene links are available in Supplementary Table 3 of McCutcheon et al. Nature Genetics, 2023. https://doi.org/10.1038/s41588-023-01554-0
SECTION: Processing and analyzing scRNA-seq
30. Upregulated DEGs were input into EnrichR’s GO Biological Process 2021 database71 for functional annotation.