GSM4261779: CA1_deep_control_replicate3; Rattus norvegicus; RNA-Seq
Brains from 3 control and 3 epileptic rats were dissected, longitudinally cut in half (separating both hemispheres), wrapped in aluminum foil and immediately frozen in liquid nitrogen. All samples were conserved at -80ºC until use. The hippocampal region of each hemisphere was cut in 20μm slices in a cryostat (Leica) (chamber temperature: -20ºC; block temperature: -30ºC) and placed on 1.0mm PEN-membrane covered slides (Carl Zeiss). Slides were conserved at -20ºC until use. Right before microdissection, slides were dried with vapor of liquid nitrogen. CA1 pyramidal cell layer was microdissected with a Leica 6000 laser microdissector through a 40x non-oil immersion objective to obtain cell bodies of superficial and deep sublayers separately. Microdissected deep and superficial areas were collected in different empty caps of 0.5ml Eppendorf tubes. After microdissection, samples were processed following ARCTURUS PicoPure RNA Isolation Kit (Thermo Fisher Scientific) instructions in order to extract and isolate total RNA. Briefly, 50μl of extraction buffer was added into the cap, incubated at 42ºC for 30min, centrifuged at 800xg for 2min and stored at -80ºC. The same volume of 70% ethanol was added to the cell extract and the mixture was pipetted into a pre-conditioned RNA purification column. The column was centrifuged 2min at 100xg and 30sec at 16000xg, and washed with 100μl of Wash Buffer 1. To completely eliminate DNA, the purification column was treated with 40μl of DNase (diluted 1/8 in RDD Buffer) (Qiagen), incubated 15min and centrifuged at 8000xg for 15sec. Then the column was washed twice with 100μl of Wash Buffer 2, and centrifuged at 8000xg after the first wash and at 16000xg after the second one. Finally, RNA was eluted into a new 0.5μl Eppendorf tube by adding 11μl of elution buffer onto the column membrane, incubating the column for 1min at room temperature, and centrifuging the column for 1min at 1000xg and at 16000xg immediately after. Total RNA samples were stored at -80ºC. RNA integrity number (RIN) was similar in control (4.7 ± 0.8) and epileptic rats (5.7 ± 0.5; P=0.07; 3 replicates x 2 sublayers per group), as well as for deep (5.2 ± 0.9) and superficial sublayers (5.3 ± 0.; P=0.81; n=6 replicates per sublayers) when assayed on an Agilent 2100 Bioanalyzer. RNA-seq libraries were produced (TruSeq Stranded mRNA Sample Preparation Kit) and sequenced (HiSeq Sequencing Kit TruSeq v4) according to manufacturer instructions in a HiSeq2500 sequencer (Illumina, Inc). Libraries were strand specific (reverse) and sequencing was performed in paired-end configuration with a read length of 75bp. Library size of read pairs for the different samples analyzed was between 47 and 59 Million reads.
GEO Accession
GSM4261779