title: GSM8889691: A375, si-FOSL1, #1; Homo sapiens; RNA-Seq
library_construction_protocol: RNAseq: RNA extracted from A375 and 1205Lu cells was quantified with a Qubit Fluorometer (ThermoFisher Scientific, Waltham, MA) and screened for quality on the Agilent TapeStation 4200 (Agilent Technologies, Santa Clara, CA). Cut&Run: Cut&Run was performed with A375 and 1205Lu cells using CUTANA ChIC/CUT&RUN Kit version 4.0 (EpiCypher, Cat. # SKU: 14–1048). Briefly, 500,000 cells were collected and re-suspended in Wash Buffer and mixed with 10 μl activated ConA beads. 0.5 μg FRA1 antibody (Abcam, Cat. # ab252421) or 1 μl IgG antibody were added and incubated overnight at 4°C. Beads were washed twice in 200 μl Cell Permeabilization Buffer (CPB) prior to the addition of 2.5 μl CUTANA pAG-MNase in 50 μl of CPB per reaction. Beads were again washed twice in 200 μl CPB and suspended in 50 μl CPB. For chromatin digestion, 1 μl of 100 mM CaCl2 was added to activate the MNase. 33 μl of Stop Master Mix was added to terminate the MNase activity after 2h incubation at 4°C. To isolate DNA from the supernatant, 119 μl SPRlselect were added and incubated for 5 minutes at room temperature. SPRIselect beads were washed twice with 85% Ethanol and air-dried. To elute DNA, 17 μl of 0.1X TE Buffer were added. RNAseq: The samples were then processed for RNA sequencing using the NuGEN Universal RNA-Seq Library Preparation Kit with NuQuant (Tecan Genomics, Redwood City, CA). Briefly, 100 ng of RNA were used to generate cDNA and a strand-specific library following the manufacturer's protocol. Quality control steps were performed, including TapeStation size assessment and quantification using the Kapa Library Quantification Kit (Roche, Wilmington, MA). The final libraries were normalized, denatured, and sequenced on the Illumina NovaSeq 6000 sequencer with the S1–200 reagent kit to generate approximately 60 million 100-base read pairs per sample (Illumina, Inc., San Diego, CA). Cut&Run:  Eluted DNA was quantified with the Qubit fluorometer, and 0.6 to 5 nanograms of enriched DNA was used for library preparation using the Kapa HyperPrep Kit (Roche, Cat. # 07962347001) following EpiCypher's parameters for indexing PCR and library amplification as described in the CUT&RUN Library Prep Manual. The final libraries were Qubit quantitated and screened on the Agilent TapeStation D1000 ScreenTape (Agilent Technologies) to assess the fragment size distribution. Following final quantification using the Kapa Library Quantification Kit (Roche, Cat. # 07960140001), the libraries were sequenced on the NextSeq 500 using a Mid-output 150-cycle Kit in 2×50 configuration to generate 6–8 million read-pairs per sample (Illumina).