Supplementary methods

Hydroxymethylated DNA Immunoprecipitation (hMeDIP)

Genomic DNA was isolated from NAc tissue (n = 10 rats) by overnight Proteinase K treatment, phenol-chloroform extraction, ethanol precipitation, and RNase digestion. Subsequently, 300 µL fractions of DNA (20 ug) were sheared by ultrasonic treatment using the DiagenodeBioruptor (12 cycles, 30 s “ON”, 30 s “OFF”) to obtain a fragment size between ~200–600 bp. After denaturation (10 min at 95 °C), 5 µg DNA was then immunoprecipitated overnight at 4°C using 5 µL of rabbit polyclonal anti-5hmC antibody (Catalog # 39769;ActiveMotif,Carlsbad, CA) for hMeDIP assay in a final volume of 500 µL IP buffer (10 mM sodium phosphate (pH 7.0), 140 mMNaCl, 0.05% Triton X-100). We incubated the mixture with 80 µL of Dynabeads(Life Technologies, Carlsbad, CA) overnight at 4 °C and washed it three times with 700 µL of IP buffer. We then treated the beads with proteinase K for 3 h at 50 °C and recovered the hydroxymethylated DNA by phenol-chloroform extraction followed by ethanol precipitation. Sheared “input” DNA samples were collected prior to immunoprecipitation for subsequent comparison with immunoprecipitated DNA (Jayanthi et al., 2014).

hMeDIP sequencing and library construction

Immunoprecipitated DNA samples (n=6) were quantitated using the Qubit double-stranded DNA High-Sensitivity assay (Life Technologies, Grand Island, NY) 30 ng of the DNA was mixed with the End Repair Mix, incubated at 30oC for 30 min, and purified with 160 L of the AMPure XP Beads (Beckman Coulter, Brea, CA) and two successive washes with 80% EtOH. Samples were then dried at 37oC for 3 min. Bead-bound DNA was eluted with the Resuspension Buffer, and 15 L of the DNA supernatant solution was mixed with the A-Tailing Mix. The reaction was incubated at 37oC for 30 min and 70oC for 5 min, after which the Ligation Mix and a unique RNA Index Adapter was added to each DNA sample. Following incubation at 30oC for 10 min, the samples were mixed with the Stop Ligation Buffer and subsequently purified using 42.5 L of the AMPure XP Beads and two successive washes with 80% EtOH. Samples were then dried at 37oC for 3 min and bead-bound DNA was eluted with the resuspension buffer. To eliminate potential contamination by excess adapters, the resuspended DNA supernatant was purified once more with 50 L of the AMPure XP Beads and washed twice with 80% EtOH. Further, following elution from the beads, each ligated DNA sample was resolved on a gel using the Certified Low-range Ultra Agarose (Bio-Rad Laboratories, Hercules, CA). DNA migrating at the 250-400 basepair range was excised out of the agarose gel and extracted out of the gel fragments using the MinElute Gel Extraction Kit (Qiagen, Valencia, CA), according to the manufacturer’s instruction. Size-selected, gel-extracted DNA samples were then enriched by PCR amplification using the PCR Master Mix and the PCR Primer Cocktail using the following parameters: 98oC for 30 sec; 15 cycles of 98oC for 10sec, 60oC for 30 sec, and 72oC for 30sec; and 72oC for 5 min. Only 15 cycles were used to maximize the library diversity and to minimize amplification bias. The resulting PCR-amplified DNA libraries were purified one more time with 50 L of the AMPure XP Beads and two successive washes with 80% EtOH. Samples were then dried on at 37oC for 3 min and bead-bound library DNA was eluted with the Resuspension Buffer. One L of the eluted DNA was resolved on the 2200 TapeStation High-Sensitivity ScreenTape (Agilent, Santa Clara, CA) to verify proper basepair size (250-400) and amount. The DNA libraries were then further quantified using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA) on the Applied Biosystems 7900HT Fast Real-Time PCR System, according to the manufacturers’ instructions. DNA libraries each at 8 pM concentration were subsequently multiplexed for sequencing on the Illumina HiSeq2500 at the Johns Hopkins Next Generation Sequencing Center. The investigators performing the hMeDIP sequencing were blind to the behavioral data.

The quality of short reads was evaluated by employing FastQC program (FastQC website). FastQC uses Phred quality scores to describe the confidence of each base call in each read. Quality scores greater than 30 in per base sequence were considered as sufficient quality. Reads were then mapped on the rat genome reference sequence (rn4) by using Bowtie 1.1.1 (Langmead et al., 2009). Only uniquely aligned reads with mapping quality of ≥20 were selected for peak calling.The hMeDIP Seq dataset has been deposited in

Quantitative PCR analysis of mRNA levels

Total RNA was isolated from one hemisphere(randomly chose left or right side) ofNAc, dorsal striatum, and pre-frontal cortexusing RNeasy Mini kit (Qiagen, Valencia, CA) from 10-12 rats per group. Unpooled total RNA (0.5 μg) isolated from NAc samples was reverse-transcribed with oligo dT primers using Advantage RT-for-PCR kit (Clontech, Mountain View, CA). RT-qPCR was performed essentially as described previously (Jayanthi et al., 2005) with Roche LightCycler 480 II (Roche Diagnostics, Indianapolis, IN) using iQ SYBR Green Supermix (BioRad, Hercules, CA). For all RT-qPCR experiments, individual data were normalized using the corresponding β2-microglobulinmRNA level. The results are reported as fold changes calculated as the ratios of normalized gene expression data for METH-self-administration groups in comparison to the control group. Primers for Kcna1 (forward sequence 5’-TTG GTA AGG GTG TTC AGA AT-3’ and reverse sequence 5’-GCA AAG TAC ACT GCA CTA GA-3’), Kcna2 (forward sequence- 5’-GTG AGA GAG TGG TGA TTA AC-3’ and reverse sequence-5’-TAT CTA AGG GCA CAT TCA CAG-3’), Kcnb1 (forward sequence 5’-CTC CAT CTA CAC CAC AGC AAG T-3’ and reverse sequence-5’-CTG AAC TTG GGA CTG GTA CTC C-3’), Kcnb2 (forward sequence- 5’-GAA GAA CTT AGA AGG GAG GC-3’ and reverse sequence- 5’-GAT GAA CAG GAT AGA CAC GAT-3’),Kcnma1 (forward sequence- 5’-ACT CGT GAA CGA TAC TAA-3’ and reverse sequence- 5’-CTG TAA CCC CCT CGA A-3’), Kcnn1 (forward sequence- 5’-GCG TAA GTT CCT TCA GGC CA-3’ and reverse sequence- 5’-TAT GCG ATG CTC TGT GCC TT-3’),Kcnn2 (forward sequence- 5’-TAT GCG CTC ATC TTC GGC AT-3’ and reverse sequence- 5’-ACT GTA TTT CCC TGG CGT GG-3’)and Kcnn3 (forward sequence- 5’-GGT GAT AGA GAC CGA AC-3’ and reverse sequence- 5’-TCA GGT ATA GGC GCA A-3’) were synthesized at the Synthesis and Sequencing Facility of Johns Hopkins University (Baltimore, MD).

All the quantitative data are presented as mean + SEM. For calculating significance, one-way ANOVA followed by Fisher’s PLSD post hoc analysis was used (Stat View version 4.0).

Reference:

Jayanthi S, McCoy MT, Chen B, Britt JP, Kourrich S, Yau HJ et al. Methamphetamine downregulates striatal glutamate receptors via diverse epigenetic mechanisms. Biol Psychiatry 2014; 76: 47-56.

Jayanthi S, Deng X, Ladenheim B, McCoy MT, Cluster A, Cai NS et al.Calcineurin/NFAT-induced up-regulation of the Fas ligand/Fas death pathway is involved in methamphetamine-induced neuronal apoptosis. Proc Natl Acad Sci U S A 2005;102:868-873.

Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25.