GSM8758987: P21 Mouse retina WGBS Cre- Replicate 3; Mus musculus; Bisulfite-Seq
Retinas from P21 animals were dissected in PBS and homogenized in 1ml TRIZOL solution. 200µl of chloroform was added to homogenized TRIZOL solution, mixed by vortexing and incubated for 3 min at RT. Lysates were centrifuged for 10 min at 4C at max speed (16,000g). The aqueous phase with RNA was transferred into a fresh tube without disturbing the interphase. The rest of the volume, containing DNA and proteins was washed with 300µl 100% ethanol, inverted several times, and then centrifuged for 5 min at 4C at 2000g. The supernatant containing proteins was carefully removed so as to not disturb the precipitated DNA pellet. The DNA pellet was washed in 1mL of 100mM sodium citrate in 10% ethanol and incubated for 30 min at RT followed by centrifugation 5 min at 4ºC at 2000g and removal of the resulting supernatant. This step is repeated twice. 1.5mL of 75% ethanol was added to the DNA pellet and incubated for 20 min at RT using occasional mixing during the 20-minute incubation. The supernatant is discarded, and the DNA pellet is air dried for 10 min. 100µl of 8mM NaOH in 1mM EDTA pH 7-8 was added to resuspend the DNA pellet. After that, samples are centrifuged for 10 min at 4ºC and max speed (16,000g). The supernatant is transferred to a new tube and stored at -20ºC. DNA concentrations were measured in a Qubit fluorometer. Genomic DNA was quantified using the Qubit fluorometer. 200ng of gDNA, including 0.2% Lambda DNA (N6-methyladenine-free; NEB) was fragmented in a final volume of 50uL using the Covaris LE220 targeting ~350bp inserts. A 1.5x AMPure clean-up was utilized after fragmentation to concentrate the sample. The fragmented gDNA was bisulfite converted with the EZ-96 DNA Methylation-Gold Mag Prep Kit (Zymo Research, Cat # D5043) according to the manufacturer's recommendations. Bisulfite converted DNA was quantitated on the Qubit Fluorometer using the ssDNA Assay Kit (Thermo Fisher Scientific, Cat #Q10212). Whole genome bisulfite libraries were constructed with ~100ng of converted DNA using the xGen™ Methyl-Seq Library Prep Kit (Integrated DNA Technologies, Cat # 10009825) using unique dual indexes (IDT, 0008053) and eight PCR cycles for incorporation and amplification of indexed libraries, followed by a final 0.85x AMPure cleanup. Final libraries are quality checked for average library size and concentration. Molar concentration of libraries was determined using the KAPA Library Quantification kit (Roche Diagnostics). Libraries were sequenced on NovaSeq X using 150bp paired end reads. bACE-seq: 10-15ng of RNAse treated, TRIZOL purified gDNA was used for each sample. Samples were diluted to 10µL in MilliQ water and pre-heated by incubating at 50C for 20 minutes on a thermocycler with a heated lid set to 95ºC. 32.5µL of CT Conversion Reagent (Zymo EZ DNA Methylation Direct Kit) was then added to each sample before running the the following program on a thermocycler (98ºC for 8min, 64ºC for 105 min, followed by 98ºC for 8 min, 64ºC for 105 min), with the lid set to 95ºC. Following the bisulfite reaction, the tubes were placed at -20ºC overnight, or for 16 hours. The bisulfite converted DNA was then purified using the Zymo EZ Methylation Direct Kit, eluting with 17µL of MilliQ water for a final elution volume of 16µL. Each sample was incubated at 95ºC for 3 minutes on a thermocycler, with the lid set to 105ºC. The samples were then immediately placed into a dry-ice ethanol bath to snap cool them and were left in the cooling bath for 5 minutes. The samples were then placed on a pre-chilled PCR rack and kept on ice. 8µL of premixed APOBEC reaction mixture (2.4µL APOBEC reaction buffer (E7134A, NEB), 0.48µL APOBEC (E7133AA, NEB), 0.48µL BSA (B9000S, NEB), water to 8µL) was added to each tube and samples were incubated at 37ºC on a thermocycler with the lid set to 95ºC. After 30 min, the samples were mixed and placed back on the thermocycler for two and a half hours at 37ºC with the lid set to 95ºC. DNA was purified from the reaction using 2X Agencourt AMPure XP SPRI beads (add 2X volume of SPRI beads, incubate at room temperature for 10 minutes, place samples on magnet and wash 2 times with fresh 80% ethanol, dry for 3 min, elute in 9µL Low EDTA TE (IDT). Sequencing libraries were constructed using the xGen Adaptase Module with minor modification. 250nM of random stubby index primer was added to each sample (xGen Adaptase Module, P5L_AD002_H). Samples were incubated at 95ºC on a thermocycler with the lid set to 105ºC for 3 min, then cooled on ice for 2 min. 10µL of BST-based random priming mixture (2µL 10X ISO AMP Buffer II (B0374S, NEB), 0.8µL BST 3.0 (M0374S, NEB), 1.2µL 200mM MgCl2, 2.8µL 10mM dNTP, 3.2 µL water) was then added to each sample and incubated at 60ºC for 1 hour on a thermocycler with the lid set to 95ºC. Fragment ends were blunted and dephosphorylated by adding 2µl Exonuclease I (M0293L, NEB) and 1µl shrimp alkaline phosphatase (M0371S, NEB) to each sample. The samples were then incubated at 37ºC for 30 min and purified using a 1.6X SPRI beads (add 1.6X volume of SPRI beads, incubate at room temperature for 10 minutes, place samples on magnet and wash 2 times with fresh 80% ethanol, dry for 3 min, elute) Samples were eluted in 10 µL Low EDTA TE (IDT). In brief, samples were denatured by incubating at 95ºC on a thermocycler with the lid set to 105ºC for 3 min, then cooled on ice for 2 min. Samples were indexed and amplified using the PCR protocol in the xGen Adaptase Module using 21 cycles. The indexed samples were purified using 1.6X SPRI beads (add 1.6X volume of SPRI beads, incubate at room temperature for 10 minutes, place samples on magnet and wash 2 times with fresh 80% ethanol, dry for 3 min, elute) and eluted in 12µL Low EDTA TE (IDT).