title: GSM8828000: Control_FFPE_decalcified_MousePaws_H3K27ac; Mus musculus; OTHER
library_name: GSM8828000
library_construction_protocol: Tissue samples were cryosectionned (16 µm sections for mouse embryo tissue and  and 20 µm sections for mouse brain WT tissue), collected between -15°C and -12°C then deposited on the DNA-arrays defined regions. Tissue sections were fixed with 1% Para-formaldheyde (PFA) (Life technologies 28908) in 1X PBS (Life technologies 70011-036) at room temperature during 30 minutes, then washed three times with 1X PBS. for FFPE samples, Mouse paws have been collected and fixed in 4% Paraformaldehyde (PFA) in 1X PBS for 24 hours. Then paws have been decalcified for 7 consecutive days in  an +4°C refrigerated room on agitation. A maximum of 5 paws, each inside a cassette, are decalcified  in 1L solution of 8,5g NaCl, 10% formol and 100mL of 99-100% glacial acetic acid. At mid incubation time, decalcification solution has been renewed and paws have been cut in half and put back into the cassette. Once decalcification is finished, paws have been rinsed in ddH20 to then be dehydrated and infiltrated with paraffin using an automated device (Novalix facilities) following this standard protocol: 70% ethanol, 2 washes of 70% ethanol, 96% ethanol, 100% ethanol, half and half mix of 100% ethanol with 100% xylene, empty the incubation chamber and final step of 100% xylene. Once dehydrated, each half paw has been paraffine embedded. All blocks of mouse paw FFPE can be stored at room temperature. 4µM thick sections around the Talus bone have been collected from those FFPE mouse paw blocks using a microtome. Paw FFPE sections were deparaffined and rehydrated following standard protocols on an automated device (Novalix facilities). Tissue sections were permeabilized during 1 hour at room temperature with 1% Triton X-100 (Merck 93443) in 1X PBS solution, then washed three times with 1X PBS followed by 5 minutes incubation at room temperature with 0.1M HCl for chromatin loosening, and washed three times with 1X PBS. Afterwads, sections were covered during 1 hour at room temperature with a blocking and permeabilizing mix (10% Normal Donkey serum (NDS) (Merck D9663), 0.25% Triton X-100 (Merck 93443), 0.1% Bovine Serum Albumin (BSA) (Merck A9418), 2 mM EDTA (Life technologies AM9260G), 0.5 mM Spermidine (Life technologies 132740010), 1 tablet EDTA-free Protease Inibitor Cocktail (PIC) (Merck 11873580001)) prepared in 1X PBS. Then tissue were washed twice with 150 mM NaCl Buffer (20 mM Hepes-KOH 1M pH=7.2 (Merck 391338), 150 mM NaCl; 0.1 % BSA, 0.5 mM Spermidine, 1 tablet PIC). After that, sections were incubated with a solution containing 2 μg of a selected primary Antibody (anti-Histone H3 (tri methyl K4) antibody (Abcam ab213244); anti-Histone H3 (acethyl K27) antibody (Abcam ab177178); or anti-Histone H3 (tri methyl K27) antibody (Abcam ab 192985)), in 150 mM NaCl Buffer at room temperature during 1 hour, followed by washing twice with 150 mM NaCl Buffer. Then tissues were incubated at room temperature with a pre-incubated mix (2 hours) containing 1:1 molar ratio of unloaded fusion protein pA-Tn5 (in-house production (9.5 µM) prepared using the plasmid Addgene #124601 by following previous protocols 3,30, and the corresponding secondary Antibody (donkey anti-rabbit IgG antibody (Merck SAB3700932). The pre-incubated mix was complemented with either a conjugated Alexa-488 secondary Antibody (donkey anti-rabbit IgG antibody, Alexa Fluor 488 (Life technologies A-21206)) or a conjugated Alexa-555 secondary Antibody (donkey anti-rabbit IgG antibody, Alexa Fluor 555 (Abcam ab 150062)), added at 1/1,000 dilution. The pre-incubated mix was used in a 5 times molar excess relative to the number of moles of the MOS sequence deposited per DNA array: for 2,048 spots it corresponds to 2,56.10-12 moles; for 4,096 spots it corresponds to 5,12.10-12 moles. After 3 hours of incubation with the assembled mix, tissue sections were washed twice with 300 mM NaCl Buffer (20 mM Hepes-KOH 1M pH=7.2, 300 mM NaCl; 0.1 % BSA, 0.5 mM Spermidine, 1 tablet PIC) to remove PA-Tn5-antibodies in excess. Then, tissue sections were incubated during 5 minutes with DAPI (Life technologies 62248) 1/5,000 dilution in 1xPBS, then washed twice with PBS 1X. DNA arrays were scanned under the TRICT filter to reveal the presence of the fiducial borders as well as the physical position of tissue sections on top of the printed DNA arrays. In case of the use of the conjugated Alexa-488 secondary Antibody, an additionnal scanning using FITC filter was done to reveal the localisation of the studied modification of histone. Finally, an additional scanning of the tissue using DAPI filter was done to observe nuclear localisation and to get cell density information. After imaging, sections were incubated overnight at 37°C with 10 mM MgCl2 (Life technologies AM9530G) in 300 mM NaCl Buffer for in situ tagmentation. Next day, tissue sections were incubated with a mix containing 2.25 µL of EDTA 0.5 M, 2.75 µL of 10% Sodium dodecyl sulfate (SDS) (Merck 71736) and 0.5 µL of 20 mg/mL Proteinase K (Life technologies 25530049) in 50 µL of ddH2O overnight at 37 °C. The mix volume was ajusted depending of the slide printing area. Finally, slides were washed under agitation (300 rpm) in containers containing: 100 mL of preheated buffer 1 (2X SSC and 0.1% SDS) during 15 minutes at 50°C, then 10 minutes with buffer 2 (0.2X SSC) at room temperature and 10 minutres with buffer 3 (0.1X SSC) at room temperature. At last the slides were washed in ddH2O and spin-dried. After that, DNA arrays were incubated with 0.1 M NaOH solution during 10 minutes at room temperature and then neutralized after removing liquid with a mix containig 100 µL of 0.1 M NaOH, 11.8 µL of 10X TE and 6.5 µL of 1.25 M acetic acid during 2-3 minutes. Finally, slides were washed within a falcon containing buffer 3 (0.1X SSC), then into a falcon containing ddH2O and spin-dried. DNA arrays were incubated with a poly-C tailing mix containing 0.6 U/µL terminal transferase (New England Biolabs M0315), 1X terminal transferase buffer (New England Biolabs BO315), 0.3X CoCl2 (New England Biolabs B0252), 0.2 mM dCTP and 0.01 mM ddCTP during 35 minutes at 37°C, then 20 minutes at 70°C, and cooled at 12°C. To avoid evaporation, each printing area was closed by using sealing chambers (Life technologies AB-0576 (size 25 μL) or AB-0577 (size 65 μL)). Finally, slides were washed within a falcon containing buffer 3, and a falcon containing ddH2O, prior spin-dry. DNA arrays were then incubated with a mix containing:  0.1 U/µL  Klenow exo- (New England Biolabs M0212), 0.2 mg/mL BSA, 1X NEB2 buffer (New England Biolabs B7002), 0.5 mM dNTPs and an oligonucleotide (1 µM) having a complementary sequence for the poly-C tailed sequence, as well as a 5'-extremity providing an adapter sequence (“GTTCAGACGTGTGCTCTTCCGATCTGGGGGGGGGH”). As in the preceding step to avoid evaporation, the reaction was performed by using sealing chambers.The following conditions were used: 47°C during 5 minutes (primer annealing), 37°C during 1 hour (extension), 10 minutes at 70°C (enzyme deactivation) and cooled et 12°C. In order to recover the synthesized complementary DNA strands, DNA arrays were incubated with 100 µL of 0.1 M NaOH solution during 10 minutes, then the liquid was collected and a second incubation with 0.1 M NaOH solution was repetead. The two fractions were collected together and neutralized (for 100 µL 0.1 M NaOH solution add 11.8 µL of 10X TE and 6.5 µL of 1.25 M acetic acid). DNA arrays were also neutralized with 100 µL 0.1 M NaOH solution mixed with 11.8 µL of 10X TE and 6.5µL of 1.25 M acetic acid, then washed with buffer 3 and ddH2O and stored at 4 °C  for eventual supplementary experiments. The neutralized collected solution were precipitated with cold 100% ethanol (2X volume),  200 mM NaCl and 1 µL of 20 mg/mL glycogen (Merck 10901393001) and conserved at -20°C for at least 1 hour, then centrifuged at 12,500 rpm during 30 minutes, washed once with cold 70% ethanol and resuspended (after 30 minutes air drying) in 23 µL of ddH2O. Half of the resuspended cDNA's complementary-probe material (12ul) was adjusted to 23ul with water, then mixed with: 25 µL of Q5 hot start high fidelity 2X master mix (New England Biolabs M0464L), 1 µL of adapter seq1 primer (0.02 µM (GTTCAGACGTGTGCTCTTCCGATCT)) and 1 µL of adapter seq2 primer (0.02 µM, (TACACTCTTTCCCTACACGACGCTCTTCCGATCTGACTCGTAATAC, corresponding to a part of the T7-promoter sequence)). This mix was amplifyied by PCR  (98°C, 30 s; 15 cycles: 98°C 10 s; 65°C 75 s; then 65°C 5 min; 12°C hold) and then cleanned at 0.9X ratio with SPRIselect beads (Beckman Coulter B23318) and resuspended in 24 µL of ddH2O. A second PCR amplification was done by mixing the 24 µL of adaptors linked cDNA's complementary-probe strand with: 25 µL of Q5 hot start high fidelity 2X master mix, 0.5 µL of universal primer and 0.5 µL of index primer (0.1 µM final concentration NEBNext multiplex oligos for Illumina index primers set 1 E7335) by using the following cycling conditions: 98°C for 30 s, 15 cycles of 98°C for 10 s and 65°C for 75 s; then final extension at 65°C for 5 min and hold at 12°C. Then sample was cleanned at 0.9X ratio with SPRIselect beads. At last the library was used for Illumina sequencing (150 nts paired-ends sequencing; NovaSeq).