title: GSM5734017: PALMD+/-3; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manual instructions. For tissue samples, grind about 60mg with liquid nitrogen into powder and transfer the powder samples into the 2ml tube contains 1.5ml Trizol reagent. The mix was centrifuge at 12000×g for 5min at 4°C. The supernatant was transferred to a new 2.0ml tube which was added 0.3ml of Chloroform/isoamyl alcohol (24:1) per 1.5ml of Trizol reagent. After the mix was centrifuged at 12000×g for 10min at 4°C, the aqueous phase was transferred to a new 1.5mL tube which was add equal volume of supernatant of isopropyl alcohol. The mix was centrifuged at 12000×g for 20min at 4°C and then removed the supernatant. After washed with 1ml 75% ethanol, the RNA pellet was air-dried in the biosafety cabinet and then dissolved by add 25µL~100µL of DEPC-treated water. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).  A total amount of 2μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina ® （NR604-01/02）following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Then we added fragmentation buffer to break the mRNA into short fragments. First strand cDNA was synthesized using random hexamer primer and RNase H. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. And then, the double stranded cDNA was purified byAMPure P beads or QiaQuick PCR kit. The purified double stranded cDNA was repaired at the end, added a tail and connected to the sequencing connector, then the fragment size was selected, and finally the final cDNA library was obtained by PCR enrichment.