title: GSM9010075: mouse pulmonary microvascular endothelial cells, Co-cultured with untreated TCMK-1 cells, C2; Mus musculus; RNA-Seq
library_construction_protocol: RNA extraction and detection Reagent kit: InvitrogenTRIzol reagent Operation steps: 1. Homogenization treatment Take the sample in a 2ml grinding tube and thoroughly grind it using a grinder at 60Hz for 60 seconds. Then take 2-4 samples each time, add 1ml TRIzol reagent to the ground powder and mix immediately (the sample needs to be kept at low temperature before adding TRIzol). After adding all the samples to TRIzol, wet beat at 55Hz for 30 seconds and incubate at room temperature for 5 minutes to facilitate complete separation of nuclear protein bodies in the homogenized sample. Centrifuge at 12000rpm for 5 minutes and take the supernatant (a 1.5ml centrifuge tube can be prepared during centrifugation to hold the supernatant). 2. Phase separation Add 0.2ml chloroform to the supernatant, shake the tube vigorously for 30 seconds, and incubate at room temperature for 2-3 minutes. Centrifuge at 4 ° C and 12000rpm for 15 minutes (during centrifugation, prepare a 1.5ml centrifuge tube and write the sample name and number). The mixture separates into a red lower layer (phenol chloroform phase), an intermediate phase, and an upper colorless aqueous phase. RNA exists in the aqueous phase. 3. RNA precipitation Transfer 400 μ l of aqueous phase to a 1.5ml final tube, add 500 μ l of isopropanol, invert and mix well (about 30 times). Incubate the sample at room temperature for 10 minutes. Centrifuge at 4 ° C and 12000rpm for 10 minutes. 4. RNA washing Discard the supernatant and add 1ml of 75% ethanol to wash the precipitate (try to resuspend the precipitate as much as possible). Centrifuge at 4 ° C and 12000rpm for 5 minutes. 5. Dissolve RNA again At the end of the process, briefly centrifuge and extract ethanol, then add an appropriate amount of RNase free water-soluble sample directly. Do not vacuum centrifuge RNA. It is important not to let the RNA precipitate completely dry, as this will greatly reduce its solubility. Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.