Illumina RNA-seq of UnWinged aphid LSR1.G1.AC fragment library
RNA Fragmentation:    1. Assemble the following reaction:  10 x Fragmentation Buffer [Ambion AM8740]  1 µL  RNA Transcriptome(less then100 ng Total)   8 µL    Ambion Spike-In Controls (see additional Protocol) 1 µL  2. Incubate tube in a PCR thermocycler at 70°C for exactly 3 min 50 sec., add 1 µL of Stop Buffer, and put the tube on ice.  3. Bring volume up to 50 µL by adding 39 µL DEPC-H2O and clean with Zymo RNA Clean and Concentrate-5 kit [Zymo R1015] and elute in 10 µL DEPC-H2O.  cDNA synthesis:    First Strand  1. Assemble the following reaction in a 200 µL thin wall PCR tube.  Random Hexamer Primers (3 µg/µL) [Invitrogen 48190-011]     1 µL  Oligo(dT)20 Primer (0.3 µg/µL) [Invitrogen 18418-020]  0.5 µL  RNA Transcriptome(less then100 ng)    8.5 µL  2. Incubate in a PCR thermocycler at 65°C for 5 min, and then place the tube on ice.  3. Mix the following in order listed, make master mix for multiple samples:  5x First Stand Buffer    4 µL  100 mM DTT     2 µL  10mM dNTP Mix     1 µL  Superase-IN (20 U/µL) [Ambion AM2694]   1 µL  4. Add 8 µL of mixture to the tube, mix well, and heat at 25°C in a thermocycler for 2 min.  5. Add 1 µL SuperScript II (200 U/µL) [Invitrogen 18064-014] and incubate in PCR thermocycler using the following program:  Step 1 25°C 10 min  Step 2 42°C 50 min  Step 3 70°C 15 min  Step 4 4°C Hold    Second Strand   1. Place tubes from first stand cDNA synthesis on ice.  2. Add 53.5 µL of H2O to the first strand cDNA synthesis reaction.  3. Add the following reagents:  5x Second Strand Buffer [Invitrogen 18012-014]  20 µL  10mM dNTP Mix       3 µL  4. Mix well, incubate on ice for 5 min, and add the following:  RNase H (2 U/µL) [Invitrogen 18021-014]     1 µL  DNA Pol I (10 U/µL) [Invitrogen 18010-025]      2.5 µL  5. Mix well, and incubate at 16°C in thermocycler for 2.5 hrs.  6. Purify the DNA with Zymo DNA Clean and Concentrate Kit-5 [Zymo D4003] (Use 5:1 Ratio in Zymo Protocol here and in all additional Cleaning steps).  Elute in 12 µL H2O.    End Repair:    Epicentre End-It Repair Kit [Epicentre ER0720]    1. Prepare the following reaction mix:  Eluted DNA      10 µL  H2O      24 µL  10x End-Repair Buffer    5 µL  dNTP Mix      5 µL  ATP      5 µL  End-Repair Enzyme Mix    1 µL  2. Incubate at 25°C for 45 min.  3. Purify the DNA with Zymo DNA Clean and Concentrate-5 kit.  Elute in 12 µL H2O.    A-Tailing:    1. Prepare the following reaction mix:  Eluted DNA      10 µL  H2O      31 µL  10x Klenow Buffer     5 µL  10mM dATP     1 µL  Klenow (3’ to 5’, exo-) (5 U/µL) [NEB M0212]  3 µL  2. Incubate at 37°C for 30 min.  3. Purify the DNA with Zymo DNA Clean and Concentrate-5 kit.  Elute in 12 µL H2O.    Ligation:    Epicentre Fast-Link DNA Ligation Kit [Epicentre LK11025]  1. Prepare the following reaction mix:  Eluted DNA      10 µL  H2O      3 µL  10x Fast-Link Ligation Buffer   2 µL  ATP (10mM)     2 µL  Adaptor Oligo (49 µM)    2 µL  Fast-Link DNA Ligase    1 µL  2. Incubate at 25°C for 15 min.  3. Purify the DNA with Zymo DNA Clean and Concentrate-5 kit.  Elute in 12 µL H2O.    PCR Enrichment of Purified cDNA Templates:    1. Prepare PCR Reaction as follows:  5x cloned Phusion HF Buffer  10 µL  Multi/Std PCR Primer 1.0 (25 µM)  1 µL  Multiplexing PCR Primer 2.0 (0.5 µM) 1 µL  PCR Primer Index # (25 µM)  1 µL  10mM dNTP Mix    1.5 µL  Phusion Polymerase [NEB F-530]  1 µL  H2O     24.5 µL  2. Add 10 µL of Gel Purified Fragments to PCR tube.  3. Run following PCR cycle:  98°C 1 min  98°C  10 sec  65°C 30 sec  18x  72°C 45 sec  72°C 5 min  4°C  hold  4. Proceed directly to next step.      Gel Purification of PCR Product    Size fractionate samples on 1.8% agarose gel (1x TBE). [17 ml 10x TBE, 153 ml dH2O , and 3.06 g Agarose]     Cut out band that is approximately 250-550 bp.    Purify DNA with Zymo Zymoclean Gel DNA Recovery.  Do not incubate any higher than 37°C. Elute in 27 µL EB.            Quantify on QuBit fluorometer