title: GSM1190973: B6 9hr rep2; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized by cervical dislocation. Eyes were enucleated and immediately placed in RNAlater stabilization reagent (Qiagen, Valencia, CA, USA) to preserve RNA content and integrity for whole eye runs. One mouse eye or two retinas were homogenized at once and passed through a QIAShredder column (Qiagen) as per manufacturer’s directions to further homogenize the eye tissues. Total RNA was then purified by using the RNeasy Mini Kit (Qiagen) with on column DNase treatment (Qiagen) as per manufacturer’s directions. Poly(A) RNA was isolated with the Oligotex kit (Qiagen) as per the manufacturer’s instructions. Pooled total RNA samples of 5 female mice 4 weeks of age were used for each whole eye library preparation.  For first strand cDNA synthesis, instructions from the SuperScript III kit protocol (Invitrogen) were followed. About 400-450 ng of isolated poly(A) RNA was mixed with 50 ng of random primers and 1 mM deoxyribonucleotide triphosphate (dNTP), incubated at 65°C for 5 min and then placed on ice for 5 min. A reaction mixture comprising 5 mM MgCl2, 10 mM DTT, 40 U RNaseOUT, and 200 U SuperScript III reverse transcriptase was added to the initial mix to achieve a total volume of 20 µL. The mixture was incubated at 25°C for 10 min, followed by 50 min at 50°C. The reaction was terminated at 85°C for 5 min and then chilled on ice for 10 min. At this point, 2 U RNase H was added and the mix was incubated at 37°C for 20 min. The first strand cDNA synthesis reaction was immediately used for second strand synthesis. To the first strand product, 300 µM dNTP, E. coli DNA polymerase I buffer, and water was added to obtain a total volume of 95 µL and allowed to incubate on ice for 10 min. Then, 0.05 U E. coli DNA polymerase I (New England Biolabs, Beverly, MA, USA) was added and the mixture was incubated at 16°C for 2.5 h. The resulting double stranded cDNA was purified with the Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA) and eluted in 100 µL nuclease-free water and then fragmented by the Covaris S2 instrument (Woburn, MA, USA) to generate ~200 bp fragments as follows: 10% duty cycle, intensity of 5, 100 cycles per burst, with a bath temperature of 7.7°C and an acoustic power of 24 W.   The Illumina library was prepared according to the manufacturer’s instructions and purified using the Wizard SV Gel and PCR Clean-up System (Promega). Overhangs were converted into blunt ends with T4 DNA polymerase and Klenow DNA polymerase by incubating the mixed sample at 20°C for 30 min. cDNA was purified and eluted in 32 µL of nuclease free water with the Wizard Plus Minipreps DNA purification system (Promega). The purified sample was then mixed with Klenow fragment (3’ to 5’ exo minus) and incubated at 37°C for 30 min to add an ‘A’ base to the 3’ end of the blunt phosphorylated DNA fragments. The cDNA was then purified and eluted in 23 µL of nuclease free water with the Wizard Plus Minipreps DNA purification system (Promega). Eluted DNA was mixed with Illumina Adapter Oligo mix and T4 DNA ligase and incubated at room temperature for 15 min to ligate adapters to the ends of the DNA fragments to prepare them for hybridization to the flow cell. cDNA then was purified and eluted in 10 µL of nuclease free water with the Wizard Plus Minipreps DNA purification system (Promega). cDNAtemplates were purified by running samples on a 1% agarose gel at 120 V for 60 min and excising the region of the gel in the 200 bp range. The 200 bp cDNA enriched fragments were purified and eluted in 30 µL of nuclease free water with the Wizard PlusMinipreps DNA purification system (Promega). cDNA in the library was then amplified by a 15 cycle PCR with two primers that annealed to the ends of the adapters. The amplified cDNA was purified and eluted in 30 µL of nuclease free water with the Wizard Plus Minipreps DNA purification system (Promega). The size, purity and concentration of the final library was checked with the Bio-Rad Experion DNA specific chip prior to sequencing by using the Illumina Genome Analyzer IIx.
attributes.tag: GEO Accession
attributes.value: GSM1190973