title: GSM7519415: ChIP_H3k4me3_WT, rep1; Mus musculus; ChIP-Seq
library_construction_protocol: For RNA, total RNA was extracted from hippocampal tissue with TRI reagent (Sigma-Aldrich) as recommended by the manufacturer.  Subsequently, genomic DNA was eliminated by a treatment with DNAse I (Qiagen) for 30 min at 25ºC. Followed by precipitation with phenol-chloroform-isoamyl alcohol (Sigma-Aldrich). ChIPs were performed as previously described in detail (Galvão-Ferreira et al., 2017) with minor modifications. Briefly, 3 μg of antibody were incubated with Dynabeads coated to Protein G (Invitrogen) in RIPA-150 buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM, EDTA, 0.1% SDS, 1% Triton X-100, and 0.1% sodium deoxycholate; pH 8) for 6-18 h at 4°C in agitation. Nuclei from hippocampal cells were extracted in a dounce homogenizer as explained for the FANS method. Samples were fixed with 1% formaldehyde (Sigma-Aldrich) for 10 min at room temperature followed by 0.1 M glycine for 5 min to stop the fixation. Nuclei were centrifuged at 1,5 G, and the pellet was resuspended in 100 μL of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris; pH 8) prior to its sonication in a Bioruptor Pico (Diagenode) for 12 cycles of 15 s on/30 s off. After 6 min of centrifugation at 13000 rpm, the supernatant containing isolated chromatin was collected. Samples were diluted in 900 μL of ChIP Dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl; pH 8) and incubated overnight at 4°C with the antibody-Dynabeads mix. Beads were thoroughly rinsed at 4ºC as follows: two washes in RIPA-150 buffer, three washes in RIPA-500 buffer (50mM Tris-HCl, 500 mM NaCl, 1 mM, EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate; pH 8), two washes in RIPA LiCl buffer (50 mM Tris-HCl, 1 mM EDTA, 1% NP-40, 0.7%, sodium deosycholate, 500 mM LiCl2), and two final washes in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA; pH 8.0), five minutes each. Samples were resuspended in 200 μl Elution buffer (10 mM Tris-HCl, 5 mM EDTA, 300 mM NaCl, 0.5% SDS; pH 8) and treated with 1 μl of 10 mg/mL RNase A (Fermentas) overnight at 65°C in agitation for crosslink reversion. Samples were treated with 3 μl of 20 mg/mL Proteinase K (Thermo Scientific) for 2 hours at 55°C in agitation. Last, DNA precipitation was performed by phenol-chloroform-isoamyl alcohol (Sigma-Aldrich). For Kdm1a ChIP, some modification were applied as in (Lipinski et al., 2020). In brief, samples required a stronger fixation treatment in 1% PFA for 30 min at 37ºC. To fragment these highly fixed samples, 43 cycles of sonication (30 s On, 30 s Off) were applied in a Bioruptor Pico (Diagenode). SDS lysis buffer was modified to RIPA buffer (0.1% SDS, 1% IGEPAL, and 0.5% sodium deoxycholate). Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) using a CTCF-specific antibody (Abclonal Cat # ab70303) was performed as described in the in situ ChIA-PET protocol (Wang et al., 2021) on approximately 10 million nuclei of hippocampal excitatory neurons sorted by FANS as described above. 4C-seq, was performed as previously described (Krijger et al., 2020) with minimal changes. Approximately 5 million hippocampal nuclei were fixed at 1 % with PFA. The restriction enzyme cutting order was Dpn II as the primary enzyme and Csp6I as the secondary enzyme. Ligated chromatin was phenol-chloroform extracted and ethanol precipitated. Reactions were then further purified with Ampure XP beads with a 0.8x ratio of bead solution to library following the manufactures instructions. Samples were then quantified with Qubit and 4C-seq libraries were sequenced using an Illumina high-throughput sequencing equip. For RNA three mice were used per genotype (Kdm1a-ifKO and their control littermates) and Poly-A libraries were generated. For RNA and ChIPseq,  libraries were generated for each independent sample and single-end sequenced in a HiSeq 2500 apparatus from Illumina. For ChIAPet, librearies were generated and paired end and 150 pb were sequenced and emrged from Miseq, Nextseq 500/550 and Hiseq 3000/4000 aparatus. PolyA RNAseq, ChIP-seq, 4C-seq and ChIAPet