DOI: 10.5061/dryad.dfn2z35fm
title: 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

description: 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&Tag)-seq revealed that high stiffness and
 fast stress relaxing groups were enriched for Sp1 target gene expression
 as well as increased Sp1 binding at genomic loci. Notably, analysis of
 publicly available claudin-low breast cancer data showed that high
 expression of the Sp1-regulated genes in fast stress relaxing groups was
 correlated with significantly reduced patient survival. Mechanistically,
 we found that phosphorylated Sp1 (T453) exhibited increased nuclear
 localization in matrices with high stiffness and fast stress relaxation.
 Furthermore, Sp1 phosphorylation was regulated by PI3K and ERK1/2
 activity, as well as actomyosin contractility. Our tunable hydrogel
 platform reveals that multiple tumor-mimicking cues within complex
 viscoelastic microenvironments reinforce malignant traits, with Sp1 acting
 as a mechanoresponsive transcription factor that transduces these signals.
 RNA-seq dataset for MDA-MB-231 and MCF-10A cells encapsulated for 7 days
 in soft or stiff, slow or fast relaxing, and low or high collagen density
 alginate-collagen matrices. The RNA-seq dataset has 8 total mechanical
 conditions containing 3 replicates per condition for MDA-MB-231 cells and
 2 replicates per condition for MCF-10A cells. The dataset contains both
 fastq and raw counts (.xlsx) files.  The CUT&Tag-seq
 dataset was generated for Sp1 in MDA-MB-231 cells encapsulated
 in soft or stiff, slow or fast relaxing, low collagen density
 alginate-collagen matrices. The dataset contains has 4 total
 mechanical conditions and 2 replicates per condition. This dataset
 contains the fastq and bigwig (.bw) files. 
description: 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# EN0202), E.Coli DNA
 Pol I (Invitrogen, Cat# 18010025), E.Coli ligase (Invitrogen,
 Cat#18052-019), and the second strand buffer (Invitrogen, #18052-019).
 After cDNA cleanup using DNA beads (AMPure, Cat# A63882), in vitro
 transcription was performed using the MEGAscript T7 kit (Invitrogen, Cat#
 A57622), resulting in amplified single stranded RNA (aRNA). After aRNA
 treatment with ExoSAP-IT PCR reagent (ThermoFisher, 78200), RNA strands
 were fragmented using a fragmentation buffer containing 200 mM
 Tris-acetate (pH 8.1), 500 mM KOAc, and 150 mM MgOAc. aRNA cleanup was
 then performed using RNA-beads (AMPure, Cat#A63987). Next, aRNA was
 reverse transcribed using a RT random hex primer, and cDNA was then
 amplified using an RNA PCR primer (RPI), a uniquely indexed Illumina
 primer, and a PCR master mix (New England, Cat#M0541S). After bead cleanup
 and quality control using a bioanalyzer (Agilent), DNA libraries were
 sequenced using the NovaSeq™ 6000 and X platforms (Illumina). The raw
 reads were then mapped to the hg19 (GRCh37) reference genome.
 For CUT&Tag-seq, 250,000 cells per sample were
 washed twice using 1 ml wash buffer (also containing protease inhibitor)
 at room temperature. A Concanavalin A bead slurry was prepared by
 dissolving the beads in 1X binding buffer and separating on a magnetic
 stand. After repeating this step once, the beads were resuspended in 20 μl
 of binding buffer and then slowly added to the cell suspension and rotated
 for 10 minutes. Next, after removing the supernatant on a magnetic stand,
 cells were suspended in 50 μl of ice-cold Sp1 antibody buffer containing
 5% digitonin, protease inhibitor, and Sp1 primary antibody (Sigma, cat #
 07645, 1:50 dilution) and left on a rotator overnight at 4 °C. The next
 day, after removing the supernatant on a magnetic stand,100 μl of diluted
 anti-rabbit secondary antibody (1:100) in Dig-Wash buffer (also containing
 protease inhibitor and 5% digitonin) was added to each sample. Tubes were
 placed on a rotator for 60 minutes at room temperature. After removing the
 supernatant, cells were washed 2X using the Dig-Wash buffer. Next, the
 assembled pA-Tn5 transposome enzyme was diluted in the Dig-300 buffer
 (also containing protease inhibitor) at a 1:100 concentration and
 incubated for 60 minutes at room temperature. After washing with 1 ml
 Dig-300 buffer twice, 125 μl tagmentation buffer (also containing protease
 inhibitor and digitonin) was added to each sample and incubated for 60
 mins at 37°C. Tagmentation was then immediately stopped by adding 4.2 μl
 of 0.5 M EDTA, 1.25 μl of 10% SDS, and 1.1 μl of Proteinase K (10 mg/ml)
 to each sample and incubated at 55°C for 60 minutes. After this, the
 supernatant was collected using a magnetic stand, and 625 μl DNA
 purification binding buffer was added to each tube, and the mixture was
 transferred to a DNA column. After spinning down tubes at 17,000 g for 1
 minute, the flow through was discarded, and the column was washed at
 17,000 g, using DNA purification wash buffer (also containing 80%
 ethanol). The DNA was eluted in 35 μl of nuclease-free water. Next, PCR
 amplification was performed using the Illumina i7 and i5 indexed primers,
 dNTPs (New England Biolabs, Cat# N0447l), and PCR master mix (New England,
 Cat# M0541S). The size distribution of the libraries was quantified on an
 Agilent Bioanalyzer 2100 and Qubit fluorometer. Libraries were subjected
 to paired-end 150 bp Illumina sequencing on a Novaseq X
 platform.
description: # 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:** Metadata file containing sample names, respective fastq
 files, and raw counts (.xlsx) for RNA-seq dataset. #### File:
 raw_reads_rna_seq.zip **Description:** RNA-seq raw counts for MDA-MB-231
 and MCF-10A cells (.xlsx files). #### File: bigwig_cutandtag_mdamb231.zip
 **Description:** contains .bw cut&tag files for MDAMB231 cells. ####
 File: cutntagseq_fastq_mdamb231.zip **Description:** cut&tag .fq files
 for MDA-MB-231 cells. #### File: RNAseq_fastq_mcf10a.zip
 **Description:** .fastq files for MCF-10A cells. replicates r1 and r2 are
 pooled into one folder.  #### File: RNAseq_fastq_MDAMB231.zip
 **Description:** fastq files for MDA-MB-231 cells. replicates r1, r2, and
 r3 are in separate folders each. ## Software : CUT&Tag Data
 Processing:  Paired-end reads were aligned to the hg38 genome using
 Bowtie2 v2.3.5, followed by duplicate removal using PICARD. Primary peak
 summits were identified using MACS2 (q &lt; 0.01), overlapping peaks
 across replicates were merged, and regions were recentered and uniformly
 expanded to ± 250 bp around the summit for downstream read counting.
 Diffbind package in R was then used to build the consensus peak set and
 obtain read counts for peaks. The .bw files can be used to visualize the
 IGV plots for Sp1 binding within the genome. The software can be
 downloaded
 here: [https://igv.org/download/html/oldtempfixForDownload.html.](https://igv.org/download/html/oldtempfixForDownload.html)