Part 4:Mapping The Epigenomic And Transcriptomic Interplay During Memory Formation And Recall in The Hippocampal Engram Ensemble
Mar 22, 2022
ali.ma@wecistanche.com
RNA-seq
μl nuclease-free water), for 30 min at 37c with gentle rocking. Augmented DNA fragments were amplified and barcoded using PCR reaction (1 cycle; 5min – 72c, 30sec – 98c, 10 cycles; 15sec – 98c, 30sec – 60c, 3min – 72c). Following the manufacturer's protocol, amplified DNA was cleaned and purified using 1.8x AMPure beads (AmpureXP beads, Beckman Coulter, A63880). After bioanalyzer QC for library size and distribution, libraries were sequenced on the Illumina Nextseq 550 platform at the MIT BioMicro Center.
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ATAC-seq analysis—The raw fastq data of 40-bp paired-end sequencing reads were aligned to the mouse mm9 reference genome using Bowtie 2.0 in paired-end mode50. The Samtools collection51 was used to sort, remove PCR duplicates (rmdup), index BAM files (index), and calculate library statistics (flagstat, see Supplementary Table 13). BAM files were downsampled from properly aligned, paired and unique reads to 50M. Open chromatin peaks were analyzed using MACS252 software Differentially accessible regions (DARs) were identified by Diffbind v1.16.353 with default parameters and settings to use DESeq2 (cutoff; FDR < 0.01; fold changes > 1.5). The full matrix of normalized counts (RPKM54) was retrieved by the DiffBind package. For a generation of bigwig tracks, pileup signal files (fragment pileup per million reads) in biography format were constructed by using the MACS2 call peak function with the -B –SPMR flags. Then, normalized fold enrichment (FE) over input lambda tracks were generated using the bdgcmp function ofMACS2 with the setting - m FE. Then bedGraph files were converted to bigwig format.
IGV55 tools were used for the visualization of bigwig files. ChromHMM56 was used to establish a chromatin state model from two independent studies that utilized bulk hippocampus tissue before and after foot shock21,22 and to determine to fold enrichment ofDARs over the state model. Motif enrichment analysis57,58 and peaks annotation was performed by HOMER59 tools. All codes are available in supplementary software file RNA extraction and library preparation—Immediately follow fixation and sorting (see tissue preparation), 4 ul of protease was added to digestion buffer (RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE, Life Technologies, AM1975). Samples were incubated in heat blocks for 15 min at 50°C, then 15 min at 80°C. Next, 0.75 mL of Trizol LS (TRIzol™ LS Reagent, Thermo Fisher Scientific, 10296028) and 0.2 mL of phenol: Chloroform solution (Phenol : Chloroform: Isoamyl Alcohol,1 Phase, VWR International, K169) was added to each sample followed by vigorous shaking for 5 min at room temperature (RT). The suspension was placed into Phase lock gel heavy tubes (PLG, 5 Prime, 2302830) and spun down at 13,000rpm for 5 min. The aquatic phase with nucleic acids was extracted from the tubes to fresh Eppendorf tube with an equal amount of ethanol 100%. RNA purification was carried out by Direct-zol™ RNA MicroPrep kit (Zymo Research, R2060) for RNA purification, according to the manufacturer’s instruction. RNA was eluted (10 ul, DEPC) and then stored at −80 °C. SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian (Clontech, 635006) was used for the library preparation and amplification. 3 biological replicates (N=3) were used for library preparation. Libraries were prepared according to the manufacturer’s instructions with no extra fragmentation step (FFPE, option2, see manual), with depletion of ribosomal cDNA and final PCR amplification (15 cycles). Libraries were sequenced on the Illumina HiSeq 2000 platform at the MIT BioMicro Center.
RNA-seq analysis—The raw fastq data were aligned to the mouse mm9 reference
genome using Bowtie 2.0. The mapped reads from gene bodies (including exons and introns reads) were processed by Cufflinks 2.260 and DEseq261 with UCSC mm9 reference gene annotation to estimate transcript abundances. Differential gene expression and downstream analyses were performed using DEseq2 and custom R scripts. Relative abundance of transcripts was measured by fragments per kilobase of exon per million fragments mapped (FPKM).
For the analysis of transcriptional programs, the expression of each gene was assessed for different memory phases (Activated -early, Activated -late, Reactivated) by their log2 fold change (log2FC) from the basal-state. 1095 of differentially expressed genes (DEGs) passed the cutoff criteria, in which we observed at least in one comparison ofP-value of< 0.01 and log2(FC) > 1. Then, genes were unbiasedly clustered by k-mean function.
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Chromosome conformation capture (Hi-C)
Nuclei from fixed cells (see tissue preparation) were sorted into 1.5 mL Eppendorf tubes containing 500 μl fresh ice-cold lysis buffer (10mM Tris pH=8, 10mM NaCl, 0.2% Igepal CA-630, Pi and PCR grade water) and frozen on dry ice and stored at −80c until they were used. Hi-C libraries were prepared from 100,000 cells/per group (collected from two animals) in two biological replicates, using Dovetail™ Hi-C kit manual (v.1.03, Dovetail Genomics, Chicago, USA). The six Hi-C libraries were sequenced (paired-end) on Illumina Nextseq 500 platform. HOMER Hi-C pipeline was used to filter experimental artifacts such as circularization, self-ligations and PCR duplicates. Filtered reads were aligned to the reference mouse genome (mm9) and further processed using HOMER HI-C pipeline.
HOMER’s Hi-C tools were used to perform principal component analysis (PCA) on the Hi- C data to identify sub-nuclear compartments (A and B) at 50 Kb resolution (see supplementary software). For compartment switch analysis, we first subtracted the PC1 values between the groups. Next we identified the top BtoA transitions as regions with PC1 change above the 90th percentile and the top AtoB transitions as regions with PC1 change below the 10th percentile. Since sub-nuclear compartments are regulated as units of several hundred kilobases, we consolidated consecutive 50 Kb AtoB or BtoA segments that were within a distance of 100 Kb of each other. Consolidated domains that were less than 200 Kb in total size we discarded. Compartment switch was only considered if a negative value was transformed to a positive value (and vice versa). Finally, positive and negative values ofthe first component were compared between all three populations of neuron (Basal, Activated - early and Activated -late).
Promoter capture Hi-C
Follow fixation and sorting (see tissue preparation) cells were sorted into 1.5 mL Eppendorf tubes containing 500 μl fresh ice-cold lysis buffer (10mM Tris pH=8, 10mM NaCl, 0.2% Igepal CA-630, Pi and PCR grade water), frozen on dry ice and stored in −80c until they were used. Hi-C libraries were prepared from 10,000 cells /per group (N=3–4), as previously described62, with some modification. Samples were spun down (4°C, 1,600 g for 20 min) and the pellet was resuspended in 400 μl of 1.2x Cutsmart buffer (New England Biolabs,
USA). First restriction digestion was carried as described in the original protocol with 1,500 U of HindIII (R3104L, New England Biolabs, USA), as the 6-base cutter (instead ofBglII in the original protocol). After Biotin labeling and overnight ligation (see original protocol for details), samples were spun down (4°C, 1,600 × g for 15 min) and the pellet was resuspended in 25 μl of PBS. The suspension was incubated in 65c with 3 μl of proteinase K for 12–16h. Purification ofHi-C DNA was carried as described in the original protocol with 100 μl of streptavidin-beads (Dynabeads M-280–streptavidin, Life Technologies, 11205D). For the second restriction enzyme, we used the 4-base cutter Hpych4V (10 U /per sample, R0620S, New England Biolabs, USA). After A-tailing reaction (see original protocol for details), samples were amplified using PCR (17 cycles) and purified with AMPure XP beads. Hi-C libraries were eluted in 20 ul of 10 mM Tris-HCl (pH 8.5). Bait capture system design for Promoter Capture Hi-C – A list of 5000 mouse gene promoters was obtained from the previously published protocol in Schoenfelder. Et al.29. The list contains only transcripts with a biotype of protein-coding regions, with HindIII restriction fragments sites. Two 120 bp capture probes were designed, one to each end ofthe fragment. The probes were synthesized by Agilent Technologies according to the SureSelect target enrichment system (Agilent Technologies).
Promoter capture Hi-C library preparation and sequencing – To capture Hi-C ligation products containing promoter sequences, we used the previously published protocol29. Briefly, hybridization blockers (Agilent Technologies) were added to the Hi-C DNA libraries. Hybridization buffer and capture bait RNA were prepared according to the manufacturer’s instructions (SureSelect Target Enrichment, Agilent Technologies). Next, the Hi-C library DNA/hybridization blockers were heated for 5 min at 95°C, before lowering the temperature to 65°C. Hi-C library DNA was mixed with hybridization buffer (prewarmed for 5 min to 65°C), and subsequently with the custom-designed capture bait system (prewarmed for 3 min to 65°C), consisting of 10,000 biotinylated RNAs targeting the HindIII restriction fragment ends of 5000 mouse gene promoters (Agilent Technologies, see Supplemental Material for capture bait design). After 24 h at 65°C in the PCR machine, biotin pulldown (MyOne Streptavidin T1 Dynabeads; Life Technologies) and washes were performed following the SureSelect Target enrichment protocol (Agilent Technologies). After the final wash, beads were resuspended in 30 μL NEBuffer 2 without prior DNA elution, and a post- capture PCR (four amplification cycles using illumine universal primers) was performed on DNA bound to the beads via biotinylated RNA. Capture Hi-C libraries were paired-end sequenced (Nextseq 500, Illumina).
Promoter capture Hi-C analysis – the HiCUP pipeline63 was employed to process the raw fastq sequencing reads. This pipeline was used to map the read pairs against the mouse (mm9) genome, to filter experimental artefacts (such as circularized reads and re-ligations) and to remove duplicate reads. For the pc-HiC data, the resulting BAM files were downsampled and processed using CHiCAGO version 1.2.064, following the default settings to call significant interactions. Full details ofthis pipeline are available from Babraham Bioinformatics
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Stereotaxic Adeno-associated Virus Injection
ArcCreERT2 mice were anesthetized with avertin and for AAV9-EF1a-DIO-hChR2-EYFP (Serotype5, obtained from UNC viral core) virus infusion. First, a small hole was made in the skull above the dorsal hippocampus (relative to Bregma: anterior-posterior −2.0 and mediolateral ± 1.5), a Hamilton syringe with a 33-gauge needle was lowered to −1.8 DV and 500 ηl of virus was infused with a flow rate of 50 ηl per minute into both hippocampi. The needle was withdrawn, following a 10 minute post-injection period and mice were allowed to recover for 4 days.
