Fig. 2: Differential junction expression in the hippocampus of chronic ethanol exposed and withdrawn rats.

Male rats were fed control liquid diet (C), ethanol liquid diet (E), or ethanol liquid diet followed by 24 h of withdrawal from ethanol diet (W). The dorsal hippocampus was dissected and RNA isolated and subjected to RNA sequencing (RNA-Seq, n = 6 per group). Differentially expressed (DE) splice junctions were identified by using STAR aligner. a Stacked bar plot depicting the upregulated, downregulated, and total number of DE junctions at FDR < 0.05. b Venn diagram of overlapping DE junctions between each pairwise comparison. c Diagram of Hapln2 gene with splice junctions represented by different letters (A, A’, B, B’, C, C’, D, E, F, and F’). d RNA-Seq average counts (reads per million) of Hapln2 junctions between C (n = 6) and W (n = 6) groups analyzed by Student’s t-test. No multiple testing correction was applied. e, f qPCR validation of junctions C and C’ for male (n = 10/group) and female rats (n = 6/group). g Identification of an intron retention event in Hapln2. The forward primer was located in exon 3 and the reverse primer in exon 4 (blue arrows). PCR was performed using male samples only and products visualized on agarose gel. The magnified portion of the Hapln2 gene diagram illustrates the location of splice junctions (C and C’) between exons 3 and 4. Agarose gel showing three bands that after sequencing were identified as containing C’ (200 bp), C (300 bp) and intron retention (500 bp). h qPCR of intron retention junction for male and female rats. Relative abundance of junction C (i), C’ (j) and intron 3 retention (k) calculated as percentage of spliced isoform (PSI) in the hippocampus of male rats. l Significant positive correlation between Pcbp1 mRNA levels and PSI of junction C’ in male rats. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01 and ****p < 0.0001 by Sidak’s test after two-way ANOVA or by Student’s t test.