TITLE: ATAC-seq of primary human fibroblasts cultured on soft and stiff ECM
AUTHORS: Brian D. Cosgrove, Lexi Bounds, Carson Key Taylor, Alan L. Su, Anthony J. Rizzo, Alejandro Barrera, Andrea R Daniel, Gregory E. Crawford, Brenton D. Hoffman, Charles A. Gersbach

[DESCRIPTION]
This protocols describes methods for Omni ATAC-seq to characterize the chromatin structural responses to changes in extracellular matrix stiffness cues. 


[STEPS]
SECTION: Cell culture and soft hydrogel processing
1. Culture primary human neonatal fibroblasts (HFF cells, ATCC CRL-2097) in DMEM with 10% FBS, 1% AntiAnti, and 1% NEAA (Sigma) on tissue culture plastic (TCP).
SECTION: Cell culture and soft hydrogel processing
2. Use polyacrylamide hydrogel 35 and 150 mm PetriSoft EasyCoat dishes (Matrigen) with an Elastic Modulus of 1 kPa (“soft”) and 50 kPa (“stiff”). 
SECTION: Cell culture and soft hydrogel processing
3. Incubate dishes for 5 minutes with sterile PBS, rinse two more times with sterile PBS, followed by the addition of 10 ug/mL fibronectin (Sigma) for 30 minutes at room temperature.
SECTION: Cell culture and soft hydrogel processing
4. Remove fibronectin and rinse dishes twice with sterile PBS, followed by a 20 minutes incubation with complete growth media.
SECTION: Cell culture and soft hydrogel processing
5. Remove media from the dishes and add cell suspensions. Allow cells to attach overnight.
SECTION: Omni ATAC-seq
6. Seed cells on Matrigen dishes of varying stiffness at slightly variable densities to account for reduced HFF attachment on softer substrates. Culture cells for 20 hours overnight. 
 
 
SECTION: Omni ATAC-seq
7. For Y-27632 ROCKi experiments, the seed cells on stiff hydrogels (as in step 6), but 1 hour prior to harvest add 10uM Y-27632 ROCKi (StemCell Tech) to the growth media of cells.
SECTION: Omni ATAC-seq
8. Use the Omni-ATAC-seq protocol to minimize mitochondrial reads from the preps (1).  
SECTION: Omni ATAC-seq
9. Following the final PCR, perform a 0.5x/1.8x double-sided SPRI clean step on the libraries. Subject libraries to quality control by determining the number of cycles required to reach 25% of the peak threshold in the diagnostic PCR. In addition, run the amplified libraries out on a High Sensitivity D1000 Tapestation (Agilent) to confirm expected size and perform Qubit dsDNA HS assays to determine a final concentration.
SECTION: Omni ATAC-seq
10. Diluted the libraries individually to 6nM and then pool them at equal volumes prior to sequencing on an Illumina HiSeq 4000 using a single lane of 50bp single end reads.
SECTION: Omni ATAC-seq
11. Use FastQC (2) to identify read quality, and trim adapter reads using Trimmomatic v0.32 (3) followed by Bowtie (4) alignment (v1.0) of the reads to the reference genome using the settings: -v 2 –best –strata -m 1 with duplicate reads removed using Picard MarkDuplicates (v1.13) and ENCODE hg38 blacklist reads removed using bedtools2 v2.25 (5). 
SECTION: Omni ATAC-seq
12. Perform peak calling using MACS2 with narrowPeak settings and a threshold of FDR < 0.001 (6), and generate a master peak set as the union set of all called peaks across every sample analyzed (224,906 unique regions total). 
SECTION: Omni ATAC-seq
13. Make count matrices using featureCounts (7) and use DEseq2 v1.36 for differential accessibility analysis (8). Perform annotation of genomic regions using ChIPSeeker (9), for interactive visualization of processed data use Degust (10) and Rstudio along with ggplot2 and tidyverse plugins to generate data visualizations. 
SECTION: Omni ATAC-seq
14. Generate sequencing-depth normalized ATAC bigWig files using deeptools bamCoverage v3.0.1 (11). Perform motif analysis using the HOMER suite (12). 
SECTION: Omni ATAC-seq
8.1. Exception to the Omni-ATAC-seq protocol: avoid trypsinization, instead perform on-plate disruption/removal of nuclei using the digitonin present in the lysis buffer. This process better preserves the nuclear mechanical context and connectivity prior to transposition by the Tn5.