title: GSM8478722: MnUS_1; Mus musculus; RNA-Seq
library_construction_protocol: 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. a. Add 5 μL rRNA Binding Buffer and 5 μL rRNA Removal Mix-Gold to each well with 10 μL total RNA (1 μg). Gently pipette the entire volume up anddown 6 times to mix thoroughly.  b. Incubate the PCR plate at 68°C for 5 min, and then place at room temperature for 1 min. c. Add 35 μL of rRNA Removal Beads to each well of the new PCR plate. Adjust the pipette to 45 μL, then with the tip of the pipette at the bottom of the well, pipette quickly up and down 20 times to mix thoroughly. Incubate at room temperature for 1 min. Place the PCR plate on the magnetic stand at room temperature for 1 min. Transfer all of the supernatant from each well to the corresponding well of the new PCR plate. d. Add 99 μL of RNAClean XP beads (If starting with degraded total RNA, add 193 μL of RNAClean XP beads to each well) to each well of the PCR plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the PCR plate at room temperature for 15 min. e. Place the PCR plate on the magnetic stand at room temperature for 5 min. Remove and discard all of the supernatant from each well of the PCR plate. f. Add 200 μL freshly prepared 70% EtOH to each well. Incubate the PCR plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. Let the PCR plate stand at room temperature for 15 min to dry. g. Add 11 μL Elution Buffer to each well of the PCR plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the PCR plate at room temperature for 2 min. Place the PCR plate on the magnetic stand at room temperature for 5 min. Transfer 8.5 μL supernatant from the PCR plate to the new 0.2 ml tube. h. Add 8.5 μL Elute, Prime, Fragment High Mix to each well of the plate. Gently pipette the entire volume up and down 6 times to mix thoroughly. i. Incubate at 94°C for 8 min, hold at 4°C, and then centrifuge shortlya. Add 8 μL First Strand Synthesis Act D Mix and SuperScript II Reverse Transcriptase in each well of the PCR plate. Gently pipette the entire volume up and down 6 times to 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. 2.3 Synthesize Second Strand cDNA  a. Add 5 μL End Repair Control (2 μL End Repair Control + 98 μL Resuspension Buffer) in each well of the PCR plate. b. Add 20 μL Second Strand Marking Master Mix. Gently pipette the entire volume up and down 6 times to mix thoroughly. c. Incubate at 16°C for 60 min. Remove the plate from the thermal cycler2.3.1 Purification  a. Add 90 μL mixed AMPure XP beads to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the plate at room temperature for 15 min. b. Place it on the magnetic stand at room temperature for 5 min. c. Remove and discard 135 μL supernatant from each well of the plate. d. With the plate on the magnetic stand, add 200 μL freshly prepared 80% EtOH to each well. Incubate the plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. e. Repeat step d one time. f. Let the plate stand at room temperature for 15 min to dry, and then remove the plate from the magnetic stand. g. Add 17.5 μL Resuspension Buffer to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. h. Incubate the plate at room temperature for 2 min. Then place the plate on the magnetic stand at room temperature for 5 min. i. Transfer 15 μL supernatant from the plate to the new PCR plate. 2.4 Adenylate 3' Ends a. Add 2.5 μL of diluted A-Tailing Control (1 μL A-Tailing Control + 99 μL 9  Resuspension Buffer) to each well of the plate.  b. Add 12.5 μL of A-Tailing Mix to each well of the plate. Gently pipette the entire volume up and down 10 times to 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. 2.5 Ligate Adapters  a. Add 2.5 μL of diluted Ligation Control (1 μL Ligation Control + 99 μL Resuspension Buffer) to each well of the plate. b. Add 2.5 μL of Ligation Mix to each well of the plate. c. Add 2.5 μL of RNA Adapter Index to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. d. Incubate at 30°C for 10 min. e. Add 5 μL of Stop Ligation Buffer to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. 2.5.1 Purification  a. Add 42 μL of mixed AMPure XP Beads to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the plate at room temperature for 15 min. b. Place the plate on the magnetic stand at room temperature for 15 min or until the liquid is clear. c. Remove and discard 79.5 μL supernatant from each well of the plate. d. With the plate on the magnetic stand, add 200 μL freshly prepared 80% EtOH to each well. Incubate the plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. e. Repeat step d one time for a total of two 80% EtOH washes. f. With the plate on the magnetic stand, let the samples air-dry at room temperature for 15 min. Remove the plate from the magnetic stand. g. Add 52.5 μL Resuspension Buffer to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. 10 h. Incubate the plate at room temperature for 2 min. Place the plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear. i. Transfer 50 μL supernatant from each well of the plate to the corresponding well of the new PCR plate. j. Add 50 μL of mixed AMPure XP Beads to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the plate at room temperature for 15 min. k. Place the plate on the magnetic stand at room temperature for 15 min or until the liquid is clear. l. Remove and discard 95 μL supernatant from each well of the plate. m. With the plate on the magnetic stand, add 200 μL freshly prepared 80% EtOH to each well. Incubate the plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. n. Repeat step m one time for a total of two 80% EtOH washes. o. With the plate on the magnetic stand, let the samples air-dry at room temperature for 15 min, and then remove the plate from the magnetic stand. p. Add 22.5 μL Resuspension Buffer to each well of the plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. q. Incubate the plate at room temperature for 2 min. Place the plate on the magnetic stand at room temperature for 5 min or until the liquid is clear. r. Transfer 20 μL supernatant from each well of the plate to the corresponding well of the new PCR plate. 2.6 Enrich DNA Fragments  a. Add 5 μL of PCR Primer Cocktail to each well of the PCR plate. b. Add 25 μL of PCR Master Mix to each well of the PCR plate. Gently pipette the entire volume up and down to mix thoroughly. c. The mixture was incubated in the following progress: 1 cycle 98°C for 30 s; 15 cycles 98°C for 10 s, 60°C for 30 s, 72°C for 30 s; 1 cycle 72°C for 5 min; hold at 10°C. 11 2.7.1 Purification  a. Add 50 μL of the mixed AMPure XP Beads to each well of the PCR plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. Incubate the PCR plate at room temperature for 15 min. b. Place the plate on the magnetic stand at room temperature for 15 min or until the liquid is clear. c. Remove and discard 95 μL supernatant from each well of the PCR plate. d. With the PCR plate on the magnetic stand, add 200 μL freshly prepared 80% EtOH to each well. Incubate the PCR plate at room temperature for 30 s, and then remove and discard all of the supernatant from each well. e. Repeat step d one time for a total of two 80% EtOH washes. f. With the PCR plate on the magnetic stand, let the samples air-dry at room temperature for 15 min, and then remove the plate from the magnetic stand. g. Add 32.5 μL Resuspension Buffer to each well of the PCR plate. Gently pipette the entire volume up and down 10 times to mix thoroughly. h. Incubate the PCR plate at room temperature for 2 min. Place the PCR plate on the magnetic stand at room temperature for 5 min or until the liquid is clear. i. Transfer 30 μL supernatant from each well of the PCR plate to the corresponding well of the new PCR plate. 2.8 Validate library  a. Load 1 μL sample on an Agilent Technologies 2100 Bioanalyze. b. Check the size and purity of the sample