GSM8185314: Batch1_PVR_PTZ_Rep4; Mus musculus; RNA-Seq
Isolation of mRNA with RiboTag: Cortices of p60 PvalbCre/RiboTag and SstCre/RiboTag mice were dissected in ice-cold PBS and homogenized in the buffer containing 100 mM KCl, 50 mM Tris-HCl pH 7.4, 12 mM MgCl2, 100 g/ml cycloheximide (Sigma), 1 mg/ml heparin (Sigma), 1x complete mini, EDTA-free protease inhibitor cocktail (Roche), 200 units/ml RNasin© plus inhibitor (Promega) and 1 mM DTT (Sigma) (500 µl ~3% w/v). The lysate was centrifuged at 2,000g for 10 minutes at 4°C. Igepal-CA380 was then added to the supernatant at the final concentration of 1%. The lysate was briefly incubated on ice and then centrifuged at 12,000g for 10 minutes at 4°C. 25 µl of the supernatant was collected as input sample. 30 µl/ml of anti-HA coupled magnetic beads (Pierce) were then added to the remaining supernatant. Incubation was performed on a rotator for 3 hours at 4°C. The beads were washed four times in the high salt buffer containing 350 mM KCl, 1% Igepal-CA380, 50 mM Tris-HCl, pH7.4, 12 mM MgCl2, 100 µg/ml Cycloheximide (Sigma) and 1 mM DTT (Sigma). Ribosomes were eluted in 350 µl of RLT plus buffer (Qiagen). RNA purification was performed using RNeasy Plus Mini kit (Qiagen) following the manufacturers' instructions. For qPCR, 20 ng of total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR quality controls were performed with the following primers:                                                                                                                                                                       Fos Forward: 5′- GGGACAGCCTTTCCTACTACC-3′ Reverse: 5′- AGATCTGCGCAAAAGTCCTG-3′ Nr4a1 Forward: 5′- TTGAGTTCGGCAAGCCTACC-3′ Reverse: 5′- GTGTACCCGTCCATGAAGGTG-3′ Gapdh Forward: 5′- TCAACGGGAAGCCCATCA-3′ Reverse: 5′- CTCGTGGTTCACACCCATCA-3′ Syt2 Forward: 5′- AGAACCTGGGCAAATTGCAGT-3′ Reverse: 5′- CCTAACTCCTGGTATGGCACC-3′ Pvalb Forward: 5′- CATTGAGGAGGATGAGCTG-3′ Reverse: 5′- AGTGGAGAATTCTTCAACCC-3′ Lhx6 Forward: 5′- CTACTTCAGCCGATTTGGAACC-3′ Reverse: 5′- GCAAAGCACTTTCTCCTCAACG-3′ RNA-seq was performed at the TSRI Next Generation Sequencing Core on the Illumina HiSeq platform. The libraries were generated, barcoded, and sequenced according to the manufacturers' recommendations. Sequencing libraries were prepared using NEBNext® Ultra™ II RNA Library Prep Kit for Illumina®