GSM8886375: MethylC-seq, pyramidal neurons, P0, Poly IC, rep1; Mus musculus; Bisulfite-Seq
Cortex dissecxtion and nuclei isolation: The frontal cortex tissue was rapidly dissected from perinatal mice on ice. The INTACT method was used to label excitatory neurons and fluoreescence-activated nuclei sorting (FANS) was used to affinity purify GFP+ nuclei. RNA-seq: RNA was extracted by using the RNeasy Mini kit with DNAse I treatment (Qiagen, 74104) for whole frontal cortex, or by using Single-Cell RNA Purification Kit (Norgen, 51800) for nuclei. Bisulfite-Seq (MethylC-seq):  Isolated nuclei were resuspended in PBS for DNA purification using the DNeasy Blood and Tissue kit (Qiagen 69504) following the standard kit protocol. RNA-seq: For whole tissue RNA, strand-specific cDNA libraries were constructed by selecting poly-adenylated mRNA transcripts using Truseq Stranded mRNA High Throughput Library Prep kit (Illumina, 20015963) with TruSeq RNA CD Indexes (Illumina, 20015949). Libraries were sequenced using Illumina NextSeq500, generating 20 million reads (75 bp, paired-end sequencing, whole cortex). INTACT-purified nuclear RNA (Norgen, 51800) was converted to cDNA and amplified with the Ovation RNA-seq System V2 (Nugen, 7102-A01) and libraries prepared with Kapa LTP library preparation kit (Illumina, KK8232) with TruSeq DNA Single Indexes (Illumina, 20015960), generating 40 million reads (150 bp, paired-end sequencing using Illumina HiSeq 4000 or NovaSeq 6000). Bisulfite-Seq (MethylC-seq):  MethylC-seq libraries were constructed using the snmC-seq2 adapted for Bulk samples (https://dx.doi.org/10.17504/protocols.io.pjvdkn6). 40ng of DNA samples  were subjected to sodium bisulfite conversion using Zymo EZ DNA Methylation-Direct Kit (D5020, D5021), 0.5% of Lambda was added to each sample, resuspended in 9ul of EB buffer, 1ul of random primer was added to each of the samples, denature for 3 min at 95°C, put in ice for 3 min, then 10ul of Random primer was added and mixed, put in thermocycler with at 4°C 5 min,  25°C 5 min, 37°C 60 min. Exonuclease/SAP mix was added next put at 37°C 30min. Samples were subjected to one round of 0.8x SPRI beads clean up. Samples were resuspended in 10ul of EB and denature once again. Adaptase mix was added next, and put at 37°C 30min and 95°C 2min. The bisulfite-converted, adapter-ligated DNA molecules were enriched by 8 cycles of PCR with the following reaction composition: 25 µL of Kapa HiFi Hotstart, 1ul of P5 index primer (30uM), and 5ul of P7 index primer (10uM), for a final vol of 50ul. The reaction products were purified using SPRI beads. MethylC-seq libraries were sequenced on Illumina HiSeq 4000 or Novaseq 6000.