RNA-seq and CUT&Tag-seq datasets for MDA-MB-231 and MCF-10A cells from: Sp1 mechanotransduction regulates breast cancer cell invasion in engineered viscoelastic extracellular matrices

Breast cancer progression involves extensive remodeling of the extracellular matrix (ECM), including increased stiffness, altered viscoelasticity (stress relaxation), and elevated collagen levels. While in vitro experiments have revealed a role for each of these factors in individually promoting malignant behavior, their combined effects remain unclear. Here, we engineered alginate-collagen hydrogels with independently tunable stiffness, stress relaxation, and collagen density to dissect how the complex ECM environment regulates cancer cell phenotype. We show that high stiffness, fast stress relaxation, and high collagen density led to changes in cell morphology, marked by decreased roundness, and promoted spheroid invasion in both breast cancer and non-transformed mammary epithelial cells. Single cell migration speed and displacement were greatest in matrices of high stiffness, low collagen density, and slow stress relaxation. RNA-seq and Cleavage Under Targets and Tagmentation (CUT&am..., For RNA-seq, cells were extracted from alginate-collagen matrices by rocking them in Falcon tubes containing 2.5 mg/ml collagenase (Sigma, Cat#C0130) solution in PBS for 30 minutes at 37°C. After this, the tubes were centrifuged, and the supernatant was removed. Following this, the pellet was dissolved in 10 ml ice-cold EDTA (50 mM) and placed on a rotator for 10 mins. The tubes were centrifuged, and the supernatant was then removed. The cell pellet was then lysed using Trizol (Life Technologies), and RNA was extracted using the total RNA mini prep kit according to the manufacturer’s instructions (Epoch Life Sciences).
Bulk-mRNA-sequencing library prep was done using the Cel-Seq2 pipeline as described previously. Briefly, 10 ng RNA was reverse-transcribed using the CelSeq2 RT-primer, DTT (0.1 M), dNTPs (New England Biolabs, Cat# N0447l), and Superscript II reverse transcriptase (Invitrogen, Cat# 18064014), followed by second-strand synthesis using RNAseH (ThermoFisher Scientific, Cat# E..., # RNA-seq and CUT&Tag-seq datasets for MDA-MB-231 and MCF-10A cells encapsulated in alginate-collagen viscoelastic matrices.

Dataset DOI: [10.5061/dryad.dfn2z35fm](https://doi.org/10.5061/dryad.dfn2z35fm)

## Description of the data and file structure

RNA-seq of MDA-MB-231 and MCF-10A cells encapsulated in Soft/Stiff; Slow relaxing/Fast relaxing, low collagen/high collagen density alginate-based hydrogels. 3 replicates per mechanical condition for MDA-MB-231 cells and 2 replicates per mechanical condition for MCF-10A cells. r1 , r2, r3 are replicate numbers.

CUT&Tag seq of Sp1 in MDA-MB-231 cells encapsulated in Soft/Stiff; Slow relaxing/Fast relaxing, low collagen density alginate-based hydrogels. 2 replicates per mechanical condition.

### Files and variables

#### File: cut-n-tag_metadata_file.xlsx

**Description:** Metadata file containing sample names, respective fastq files, and .bw files for CUT&Tag-seq dataset.

#### File: rna-seq_metadata_file.xlsx

**Description:** Metadat...,