TITLE: ATAC-seq, primary human T cells overexpressing BATF3
AUTHORS: Andrea R Daniel

[DESCRIPTION]
This protocol describes methods for performing ATACseq on human HER2 targeted CAR T cells overexpressing BATF3 or GFP. Chromatin remodeling was assessed in acutely and chronically stimulated cells. 

[STEPS]
SECTION: Transfections for high-titer lentiviral production
1. 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: Transfections for high-titer lentiviral production
2. 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
3. Exchanged media 6 hours after transfection and collect and pool lentiviral supernatant at 24 hours and 48 hours after transfection.
SECTION: Primary human CD8+ T cell cultures
4. Isolated CD8+ T cells from individual donors were obtianed directly from vials purchased from StemCell Technologies. 
SECTION: Primary human CD8+ T cell cultures
5. Culture T cells in PRIME-XV T cell Expansion XSFM (FujiFilm) supplemented with 5% human platelet lysate (Compass Biomed), 100 U/ml penicillin and 100 μg/ml streptomycin. All media were supplemented with 100 U/ml human IL-2 (Peprotech). 
SECTION: Transduction of primary human CD8+ T cells
6. Centrifuged lentiviral supernatant at 600g for 10 min to remove cellular debris. 
SECTION: Transduction of primary human CD8+ T cells
7. Concentrate lentivirus to 50–100× the initial concentration using Lenti-X Concentrator (Takara Bio). 
SECTION: Transduction of primary human CD8+ T cells
8. 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: T cell stimulation with HER2+ tumor cells
9. HER2+ SKBR3 breast cancer cells were maintained in Dulbecco's modified Eagle medium (DMEM) GlutaMAX supplemented with 10% fetal bovine serum (FBS), 1mM sodium pyruvate, 1x MEM nonessential amino acids, 10mM HEPES, 100U/ml penicillin and 100ug/ml streptomycin.
SECTION: T cell stimulation with HER2+ tumor cells
10. Transfer 1 x 105 HER2 CAR T cells (with or without BATF3 overexpression) to a new 24-well plate with 2 x105 SKBR3 cells (1:2 E:T ratio) every 3 days (T cells are stimulated on days 3, 6, and 9).
SECTION: T cell stimulation with HER2+ tumor cells
11. T cells are removed from antigen stimulation for 2 days to recover after the final round of tumor cell stimulation before ATAC-seq on day 14 after transduction. 
SECTION: ATAC-seq
12. Sort a total of 5 × 104 transduced CD8+ T cells for Omni ATAC-seq as previously described75. See the published Omni ATAC-seq reference protocol:
https://doi.org/10.1038/protex.2017.096

SECTION: ATAC-seq
13. Libraries were sequenced on an Illumina NextSeq 2000 with paired-end 50-bp reads. Read quality was assessed with FastQC and adapters were trimmed with Trimmomatic72.
SECTION: ATAC-seq
14. Trimmed reads were aligned to the Hg38 reference genome using Bowtie76(v1.0.0) using parameters -v 2–best–strata -m 1.
SECTION: ATAC-seq
15. Reads mapping to the ENCODE hg38 blacklisted regions were removed using bedtools2 (ref. 77) intersect (v2.25.0). Duplicate reads were excluded using Picard MarkDuplicates (v1.130 (ref. 78)). 
SECTION: ATAC-seq
16. Count-per-million-normalized bigWig files were generated for visualization using deeptools bamCoverage79 (v3.0.1).
SECTION: ATAC-seq
17. Peak calling was performed using MACS2 narrowPeak80 and filtered for Padj ≤ 0.001. Peak calls were merged across samples to make a union-peak set.
SECTION: ATAC-seq
18. A count matrix containing the number of reads in peaks for each sample was generated using featureCounts73 (subread v1.4.6) and used for differential analysis in DESeq2 (ref. 68) (v.1.36). 
SECTION: ATAC-seq
19. ChIPSeeker81 was used to annotate the genomic regions and retrieve the nearest gene around each peak.
SECTION: ATAC-seq
20. HOMER (v4.11) package82 was used to find transcription factor binding motifs that contributed to changes in chromatin accessibility with BATF3 OE compared to control cells. 
SECTION: ATAC-seq
20.1. We defined the set of target differentially accessible peaks using DESeq2 (Padj < 0.05) and a background set of nondynamic regions (p value > 0.2 and |log2(fold change)| < 0.2) with all sets having a sufficiently large number of sequences. 
SECTION: ATAC-seq
20.2. Next, for each set we extracted FASTA sequences from the human reference genome (GRCh38) and ran findMotif.pl to discover motifs and compute the enrichment over background. By default, this function uses a hypergeometric distribution to score motifs to calculate enrichment p-values, controlling for differences in GC-content across target and background sets.