GSM7989974: Rat, PMLGH, C2-3; Rattus norvegicus; RNA-Seq
A. Add 600 μL Lysis/Binding Buffer to the cell or tissue lysate. Then add 30 μL miRNA Homogenate Additive to homogenate, and mix well by vortexing or inverting the tube several times. Leave the mixture on ice for 10 min. B. Add a volume of Acid-Phenol: Chloroform that is equal to the lysate volume before addition of the miRNA Homogenate Additive. Centrifuge for 5 min at maximum speed (10,000 x g) to separate the aqueous and organic phases. Carefully remove the aqueous (upper) phase without disturbing the lower phase, and transfer it to a fresh tube. C. Add 1.25 volumes of room temperature 100% ethanol to the aqueous phase. D. Pipet the lysate/ethanol mixture (from the previous step) onto the Filter Cartridge (Note: Up to 700 μL can be applied to a Filter Cartridge at a time.). Centrifuge for 30 sec at 13,000 rpm to pass the mixture through the filter. Discard the flow-through. E. Apply 350 μL miRNA Wash Solution 1 to the Filter Cartridge and centrifuge for 30 sec at 13,000 rpm. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube. F. The 10 μL DNase I and 70 μL Buffer RDD QIAGEN (#79254) were mixed. Then the mixture was added to the Filter Cartridge. Leave it at the room temperature for 15 min. G. Apply 350 μL miRNA Wash Solution 1 to the Filter Cartridge and centrifuge for 30 sec at 13,000 rpm. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube. H. Apply 500 μL Wash Solution 2/3 and centrifuge for 30 sec at 13,000 rpm. Draw it through the Filter Cartridge as in the previous step. Repeat with a second 500 μL aliquot of Wash Solution 2/3. I. Spin the assembly for 1 min to remove residual fluid from the filter. Transfer the Filter Cartridge into a fresh Collection Tube. Apply 100 μL of pre-heated (95°C) Elution Solution to the center of the filter. Leave it at the room temperature for 2 min. Spin for ~20–30 sec at maximum speed to recover the RNA. Collect the eluate (which contains the RNA) and store it at –70°C. 1. Purify and Fragment mRNA a. Add 50 μL RNA Purification Beads to each well with 50 μL total RNA (4μg) and mix thoroughly. b. Incubate at 65°C for 5 min, remove at 4°C, and then place at room temperature for 5 min. c. Place it on the magnetic stand at room temperature for 5 min. Remove and discard all of the supernatant from each well. d. Remove the magnetic stand, add 200 μL of Bead Washing Buffer in each well, and mix thoroughly. e. Place it on the magnetic stand at room temperature for 5 min. Remove and discard all of the supernatant from each well. f. Add 50 μL of Elution Buffer in each well and mix thoroughly. g. Incubate at 80°C for 2 min, remove at 25°C, and then place at room temperature. h. Add 50 μL of Beads Binding Buffer in each well and mix thoroughly. The mixture was placed at room temperature for 5 min. i. Place it on the magnetic stand at room temperature for 5 min. Remove and discard all of the supernatant from each well. j. Remove the magnetic stand, add 200 μL of Bead Washing Buffer in each well, and mix thoroughly. k. Place it on the magnetic stand at room temperature for 5 min. Remove and discard all of the supernatant from each well. l. Remove the magnetic stand, add 19.5 μL of Elute, Primer,Fragment Mix in each well and mix thoroughly. m. Incubate at 94°C for 8 min, remove at 4°C, and then centrifuge. 2. Synthesize First Strand cDNA a. Add 8 μL First Strand Synthesis Act D Mix and SuperScript II Reverse Transcriptase in each well and mix thoroughly. b. The mixture was incubated in the following progress: 25°C for 10 min, 42°C for 15 min, 70°C for 15 min, hold at 4°C. 3. Synthesize Second Strand cDNA a. Add 5 μL End Repair Control (2 μL End Repair Control + 98 μL Resuspension Buffer) in each well. b. Add 20 μL Second Strand Marking Master Mix and mix thoroughly. The mixture was incubated at 16°C for 60 min. 3.1 Purification a. Add 90 μL mixed AMPure XP beads, mix thoroughly, and then place at room temperature for 15 min. b. Place it on the magnetic stand at room temperature for 5 min. c. Remove and discard all of the supernatant from each well. d. Place the plate on the magnetic stand. Add 200 μL of 80% ethyl alcohol and place at room temperature for 30 sec. Remove and discard all of the supernatant from each well. e. Repeat the above progress: place the plate on the magnetic stand; add 200 μL of 80% ethyl alcohol and place at room temperature for 30 sec; remove and discard all of the supernatant from each well. f. Place at room temperature for 15 min for air drying. Remove the plate from the magnetic stand. g. Add 17.5 μL Resuspension Buffer and mix thoroughly. h. Incubate the mixture at room temperature for 2 min. Place it on the magnetic stand at room temperature for 5 min. i. Transfer 15 μL the supernatant from each well into the new PCR plate. 4. Adenylate 3'Ends a. Add 2.5 μL diluted A-Tailing Control (1 μL A-Tailing Control + 99 μL Resuspension Buffer) in each well. b. Add 12.5 μL A-Tailing Mix and mix thoroughly. c. The mixture was incubated in the following progress: 37°C for 30 min, 70°C for 5 min, hold at 4°C. 5. Ligate Adapters a. Add 2.5 μL diluted Ligation Control (1 μL Ligation Control + 99 μL Resuspension Buffer) in each well. b. Add 2.5 μL Ligation Mix. c. Add 2.5 μL RNA Adapter Index and mix thoroughly. d. The mixture was incubated at 30°C for 10 min. e. Add 5 μL Stop Ligation Buffer in each well and mix thoroughly. 5.1 Purification a. Add 42 μL mixed AMPure XP beads, mix thoroughly, and then place at room temperature for 15 min. b. Place it on the magnetic stand at room temperature for 15 min. c. Remove and discard 79.5 μL of the supernatant from each well. d. Place the plate on the magnetic stand. Add 200 μL of 80% ethyl alcohol and place at room temperature for 30 sec. Remove and discard all of the supernatant from each well. e. Repeat the above progress: place the plate on the magnetic stand; add 200 μL of 80% ethyl alcohol and place at room temperature for 30 sec; remove and discard all of the supernatant from each well. f. Place at room temperature for 15 min for air drying. Remove the plate from the magnetic stand. g. Add 52.5 μL Resuspension Buffer and mix thoroughly. h. Incubate the mixture at room temperature for 2 min. Place it on the magnetic stand at room temperature for 5 min. i. Transfer 50 μL the supernatant from each well into the new PCR plate. j. Add 50 μL mixed AMPure XP beads, mix thoroughly, and then place at room temperature for 15 min. k. Place it on the magnetic stand at room temperature for 15 min. l. Remove and discard 95 μL of the supernatant from each well. m. Place the plate on the magnetic stand. Add 200 μL of 80% ethyl alcohol and place at room temperature for 30 sec. Remove and discard all of the supernatant from each well. n. Repeat the above progress: place the plate on the magnetic stand; add 200 μL of 80% ethyl alcohol and place at room temperature for 30 sec; remove and discard all of the supernatant from each well. o. Place at room temperature for 15 min for air drying. Remove the plate from the magnetic stand. p. Add 22.5 μL Resuspension Buffer and mix thoroughly. q. Incubate the mixture at room temperature for 2 min. Place it on the magnetic stand at room temperature for 5 min. r. Transfer 20 μL the supernatant from each well into the new PCR plate. 6. Enrich DNA Fragments a. Add 5 μL PCR Primer Cocktail. b. Add 25 μL PCR Master Mix and mix thoroughly. c. The mixture was incubated in the following progress: 1 cycle 98°C for 30 sec; 15 cycles 98°C for 10 sec, 60°C for 30 sec, 72°C for 30 sec; 1 cycle 72°C for 5 min; hold at 10°C. 7. Purification a. Add 50 μL mixed AMPure XP beads, mix thoroughly, and then place at room temperature for 15 min. b. Place it on the magnetic stand at room temperature for 15 min. c. Remove and discard 95 μL of the supernatant from each well. d. Place the plate on the magnetic stand. Add 200 μL of 80% ethyl alcohol and place at room temperature for 30 sec. Remove and discard all of the supernatant from each well. e. Repeat the above progress: place the plate on the magnetic stand; add 200 μL of 80% ethyl alcohol and place at room temperature for 30 sec; remove and discard all of the supernatant from each well. f. Place at room temperature for 15 min for air drying. Remove the plate from the magnetic stand. g. Add 32.5 μL Resuspension Buffer and mix thoroughly. h. Incubate the mixture at room temperature for 2 min. Place it on the magnetic stand at room temperature for 5 min. i. Transfer 30 μL the supernatant from each well into the new PCR plate. 8. Validate library a. Load 1 μL sample on an Agilent 2100 Bioanalyzer. b. Check the size and purity of the library.