Name: GSM7082566
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: For 4f-SAMMY-seq, 3 million fibroblasts were detached from the culture plate by 3 min incubation in trypsin-EDTA at 37°C, 5% CO2. After two washes in cold PBS, they were resuspended in 600 μL ofCSK-Triton buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 1 mM EGTA, 300 mM Sucrose, 3 mM MgCl2, 1mM PMSF, 1 mM DTT, 0.5% Triton X-100, with protease inhibitors). After 10 min incubation on a wheel at 4°C, soluble proteins and the cytoskeletal structure were separated from the nuclei by centrifugation at 900g for 3 min at 4°C; the supernatant was labeled as S1 fraction. The pellet was then washed with an additional volume of CSK-Triton buffer, resuspended in 100 μL of CSK buffer (10 mM PIPES pH 6.8, 100 mMNaCl, 1 mM EGTA, 300 mM Sucrose, 3 mM MgCl2, 1mM PMSF, with protease inhibitors) and incubated for 60 min at 37°C with 25 U of RNase–free DNase I (Invitrogen, AM2222). To stop DNA digestion, ammonium sulphate was added in the CSK buffer to a final concentration of 250 mM. After 5 min incubation on ice, the sample was pelleted at 900g for 3 min at 4°C; the supernatant, containing digested chromatin fragments, waslabeled as S2 fraction. Afterwards, the pellet was washed with 200 μL of CSK buffer, resuspended in 100 μL of CSK-NaCl buffer (CSK buffer with NaCl final concentration increased to 2 M) and then incubated 10 min on a wheel at 4°C. At the end of the incubation, the sample was centrifuged at 2300g for 3 min at 4°C and the supernatant was labeled as S3 fraction. Finally, after two washes in 200 μL of CSK-NaCl buffer, the pellet was solubilized in 100 μL of 8M urea; the final suspension was labeled as S4 fraction. For 10kh-SAMMY-seq, 10 thousand fibroblasts were treated following 4f-SAMMY-seq protocol, halving the volume in each step and using 12.5U of DNAse I (Invitrogen, AM2222); in this way we achieved a 150 fold increase in the ratio of enzyme units per starting number of cells, while maintaining the DNase concentration unaltered. Fractions S2, S3 and S4 were diluted in TE (10 mM TrisHCl pH 7.5, 1mM EDTA pH 8.0) to a final volume of 200 μl and then incubated 90 min at 37°C with 6 μL of RNAse cocktail (Ambion, AM2286), followed by 150 min at 55° with Proteinase K (Invitrogen, AM 2548) to a final concentration of 0.2 μg/μL. Next, DNA was isolated through phenol:chloroform:isoamyl alcohol (Sigma, 77617), precipitated and resuspended in nuclease-free water. On the next day, S2 from 4f-SAMMY-seq was additionally purified using PCR DNA Purification Kit (Qiagen, 28106) and DNA fragments in this fraction were separated using AMPure XP paramagnetic beads (Beckman Coulter, A63880) to obtain S2S (< 300 bp) and S2L (> 300 bp) fractions. Beads were added to the S2 fraction in a 0.95x (v/v) ratio, so to bind fragments larger than 300bp. Magnetic separation of beads from supernatant allowed the physical separation of larger fragments (on the beads) from shorter ones (in the supernatant). Larger fragments bound on beads were then washed in 85% ethanol, resuspended in water and magnetically separated from the beads (S2L fraction). Shorter fragments in the supernatant of the first step were bound to beads by adding a further 0.85x (v/v) beads ratio to the suspension; after washing in 85% ethanol and resuspension in water, they were also detached from beads (S2S fraction). Afterwards, S2 (from 10kh-SAMMY-seq), S2L (from 4f-SAMMY-seq), S3 and S4 (from both 10kh- and 4f-SAMMY-seq) fractions were transferred to screw cap microTUBEs (Covaris, 004078) and sonicated in a Covaris M220 focused-ultrasonicator to obtain a smear of DNA fragments peaking at 200 bp (settings: water bath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50; duration: 125 sec for S2 and S2L, 175 sec for S3 and S4). Fractions were quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Q32854 ) and a Qubit 4.0 fluorometer; quality control was performed by running them on an Agilent 2100 Bioanalyzer System using the High Sensitivity DNA Kit (Agilent, 5067 – 4626). Libraries were then created from each fraction using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and the Unique Dual Index NEBNext Multiplex Oligos for Illumina (NEB, E6440S).