INIA LCM (11 Regions) CIE/AIR RNA-seq Transcript Level (Dec15)

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This dataset is open. Please contact Dr. Megan K. Mulligan ( or Dr. Robert W. Williams ( if you need more information or low-level data access.

About cases

Male C57BL/6J and DBA/2J mice (10 weeks old upon arrival) were purchased from the Jackson Laboratory and assigned to either baseline untreated control (BAS), the air control (AIR), or CIE group. Mice were individually housed with free access to food (Harland Teklad, Madison, WI) and water throughout all phases of the experiments. Body weights were recorded weekly during ethanol-drinking weeks or daily during chronic intermittent ethanol (CIE) or air exposure (detailed below). Mice were housed in a temperature- and humidity-controlled an- imal facility under a reversed 12-h light/dark cycle (lights on at 0200 h). Mice were not food- or water-deprived at any time during the study. All procedures were approved by the Institutional Ani- mal Care and Use Committee at the Medical University of South Carolina (MUSC). Brain tissue was removed at MUSC and shipped to UTHSC for laser capture microdissection (LCM).

About tissue

Whole brain tissue was sectioned at 10 mm using a Leica cryostat and mounted in series with 6e8 sections per slide onto uncharged and uncoated glass slides. Mounted sections were dehydrated in 100% methanol (90 s), 70% ethanol (1 min), 95% ethanol (1 min), 100% ethanol (1 min  2), xylene (5 min). Slides were then allowed to air dry for 10 min under a fume hood.

Series were created from distinct coronal sections (bregma po- sitions based on a C57BL/6J reference brain atlas) and individual regions were matched across section and harvested by LCM (Supplemental Fig. 2). Prelimbic (PrL) and infralimbic (ILC) cortex included a series spanning from bregma 1.98 to 1.54 mm. The accumbens core (NAc) and shell (NAs) series were collected from bregma 1.54 to 0.98 mm, and dorsolateral (DLS) and dorsomedial (DMS) striatum and bed nucleus stria terminalis (BST) were collected from bregma 0.38 to 0.10 mm. Basolateral (BLA) and central nucleus (CeA) of the amygdala series were collected from bregma 0.58 to 1.22 mm and hippocampus (CA1 and CA3) was collected from bregma 1.46 to 2.46 mm. Finally, the ventral tegmental area (VTA) and primary visual cortex (VCX) series were collected from bregma 3.28 to 3.80 mm.

Arcturus XT (Life Technologies) was used to capture 13 brain areas. The infrared laser was then used to capture the tissue onto CapSure LCM caps (Life Technologies, laser spot power set to 70 mV with a duration of 25 msec).

About platform



(Updated Dec 9, 2015 by AC and RW)

Annotation data for transcripts and genes were downloaded from ENSEMBL by Arthur Centeno, December 2015. We downloaded the entire transcript database at

The positions of transcripts and genes on the mouse assembly are version GRCm38.p4 (mm10 equivalent). However, we converted all chromosome coordinates to the mm9 assembly to be consistent with all other GN1 data sets. However, for some sequences, mostly on Chr Y and the mitochondrial genome, the values are mm10 equivalent (we had no corresponding mm9 values).

We also extracted sequence data corresponding to the transcripts whenever these data were available. However in some cases we do not have sequence data at all.

About data processing

RNA from tissue trapped in the CapSure LCM caps was extracted using the PicoPure RNA isolation kit (Life Technologies) according to the manufacturer's instructions (RNA was eluted from provided capture columns in 13.5-mL nuclease-free water). RNA quality was analyzed using a Bioanalyzer (Model 100, Agilent, Foster City, CA). Samples with an associated RNA integrity number (RIN) greater than 6 were subsequently used for RNA sequencing.


Poly-A enriched mRNA was sequenced on two platforms, ABI SOLID 550XL Wildfire (65 samples) and Ion Proton (39 samples). Read length was 50 nt for the SOLID system and the average read length for the Proton system was ~180 nt. Reads generated on the SOLID system were aligned to the mm10 reference genome using the LifeScope aligner, and BAM files were subsequently generated using custom scripts for third-party downstream analysis. For the Proton system, reads were also aligned to the mm10 (Ensemble GRCm38) reference genome using TopHat2. Settings for TopHat2 are as follows: “-p 15 -N 4 –read-gap-length 6 –read-edit-dist 8 –max-insertion-length 6 –max-deletion-length 6 –max-intron-length 300000 –b2-very-sensitive”. Alignments on both platforms were splice-aware. RSeQC-2.6.1 ( was used to generate count data based on mm10 GENECODE Basic transcript annotation (43,320 transcripts detected). We selected this annotation for greater reproducibility with existing microarray data sets and to simplify downstream analysis by limiting the number of transcript models for each gene. On average, 2.5 million and 7.8 million reads uniquely aligned to transcript models on the SOLID and Proton platforms, respectively. Data were further filtered to remove tran- scripts that had less than 1 count in 90% or more samples. After filtering, 24,597 transcripts representing 12,011 unique genes remained (Supplemental Table 1). The variance stabilizing trans- form (R package DESeq2, FitType 1⁄4 local) was applied to the count data, and transformed data were corrected by dividing by transcript length to generate log2 reads per kilobase gene model (RPK) values. The use of two different sequencing platforms was corrected using batch correction (ComBat, Supplemental Fig. 3). All data filtering, transformations, and batch correction were performed using custom R scripts. Batch-corrected and transformed log2 RPK and log2 count values are available in Supplemental Tables 2 and 3, and log2 RPK data are also available at GeneNetwork [http://www.⁄4sharinginfo&GN_ AccessionId1⁄4772; Group 1⁄4 Chronic Intermittent Ethanol, Type 1⁄4 LCM Brain Regions mRNA, Data set 1⁄4 INIA LCM (11 Regions) CIE/AIR RNA-seq Transcript Level (Dec15)].


Megan K. Mulligan, Khyobeni Mozhui, Ashutosh K. Pandey, Maren L. Smith, Suzhen Gong, Jesse Ingels, Michael F. Miles, Marcelo F. Lopez, Lu Lu, Robert W. Williams


Supplementary data related to this article can be found at http://


Thanks to NIH NIAAA and funding support was provided by INIA grants U01AA013499 and U01AA06662.