GSM8482422: C4; Ovis aries; RNA-Seq
Experimental steps of RNA extraction：（1）Weighing 15mg-30mg of sample, grinding with liquid nitrogen, adding TRK Lysis Buffer or adding TRK Lysis Buffer to homogenize with homogenizer, transferring the lysate to a homogenizing column, putting the homogenizing column into a 2mL centrifuge tube, centrifuging at room temperature of 14,000xg for 2min to remove insoluble impurities, and collecting filtrate;(2) adding 70% ethanol with the same volume into the filtrate, and vortex mixing;(3) Put the RNA binding column into a collection tube, transfer the mixed solution obtained in the second step (the mixed solution transferred each time is less than or equal to 700 L), centrifuge at room temperature of 10000xg for 1min, and discard the filtrate;(4) repeat step 3 until all the mixed solution is bound to the RNA binding column;(5) Put the RNA binding column into the collection tube, add 500µl RNA Wash Buffer I into the binding column, centrifuge at 10000xg for 30s, and discard the filtrate;(6) Put the RNA binding column back into the collection tube, add 500µl RNA Wash Buffer II into the binding column, centrifuge at 10000xg for 1min, and discard the filtrate;(7) repeat step 6;(8) Put the RNA binding column back into the collection tube, and centrifuge at 10000xg for 2 min;(9) Put the RNA binding column into a new 1.5mL centrifuge tube, add 30-70L Nuclease-Free Water into the binding column, and centrifuge at 10000xg for 2min to elute RNA;(10)Nanodrop, Qbuit and electrophoretic quality inspection. Steps for building a full-length transcription library (using ONT's kit: SQK-PCS109+SQK-PBK004): (1) Take 500 ng of Total RNA samples qualified in quality inspection, and adjust the volume to 9 ul by using Nuclease Free Water; (2) connecting a reverse transcription primer; (3). Reverse transcription reaction; (4) amplification reaction; (5) purifying with AMPure beads magnetic beads; (6) sequencing linker connection; (7) configuring a computer library; (8) On the computer: the library was Loading into the R9.4 sequencing chip, and the Prometheon Sequencer (Oxford Nano Pore Technologies, Oxford, UK) was used for sequencing for 48-72 hours.