Qiaquick pcr puri cation kit

Total RNA was extracted from white muscle with E.Z.N.A. total RNA kit II and detected with the concentration and quality. To acquire the entire transcriptome information, and to nd out the growthrelated DEGs, the muscle samples of family A were mixed with equal amount and then were divided into 2 RNA pools. PolyA mRNA was isolated by Beads with Oligo (dT) after total RNA was collected and interrupted to short fragments. Random hexamer-primer was used to synthesize the rst-strand cDNA using the Qiaquick PCR Puri cation Kit (Qiagen). The second-strand cDNA was synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively (Invitrogen). Subsequently, short fragments were puri ed, enriched for end reparation and adding polyA, connected with sequencing adapters. After that, the suitable fragments were selected using agarose gel electrophoresis for the PCR ampli cation as templates. At last, the two cDNA library could be sequenced in BGI-Shenzhen using Illumina HiSeq™ 2000.

The ampli cation was done by using primers 341F (5' CCTACGGGNGGCWGCAG -3') and 805 R (5'-GACTACHVGGGTATCTAATCC-3') for V3-V4 region of 16S rRNA gene, linked to speci c linker and adapter sequences. The details of PCR conditions were as a dual index approach performed by Fadrosh et al.
2014 [49] . Shortly, the ampli cation of pure DNA was carried out by using reaction mixture of total 25 μL volume in which, 2x 12.5 µL PCR master mix (Greentech) 2 μL DNA template, 1 μL of each V3-V4 region speci c primers were added and the total volume was raised up to 25 μL by adding nucleases free water. The PCR conditions were pre-denaturation for 5min at 95 °C, followed by denaturation of 30 cycles at 95 °C for 40 sec, annealing at 55 °C for 40 sec while extension and nal extension at 72 °C for 1 min and 5 min, respectively. The ampli ed PCR products were puri ed with the QIAquick PCR puri cation kit (QIAGEN, USA) and pooled in equimolar concentration.

PCR ampli cation was performed on 12S rRNA and 16S rRNA genes and the ITS 2 spacer using primers (Table 1) and thermocycling conditions as previously described [4, 19] . Each reaction was prepared into a nal volume of 50 µl containing; 1´-reaction buffer (670 mM Tris-HC , pH 8.8, 166 μM (NH 4 ) 2 SO 4 , 4.5 % Triton X-100, 2 mg/ml gelatin) (Bioline, Humber Road, London, UK), 0.25 mM of each dNTP, 0.25 mM each of forward and reverse primers, 1.56 U BioTaq DNA polymerase (Bioline, London, UK), 1.25 mM MgCl 2 , 32.2 µl of PCR grade water and nally 5 µl of the template DNA.
The 16S ribosomal RNA gene was ampli ed in a thermocycler (Personal Thermocycler, Biometra, Göttingen, Germany) with initial denaturation of 94 °C for 5 min followed by 30 cycles at 94 °C for 30 s, 48 °C for 45 s, 72 °C for 45 s and a nal extension at 72 °C for 7 min. Ampli cation of the ITS2 and 12S ribosomal RNA was performed using similar thermocycling conditions to those of 16S at annealing temperature of 55 °C and 52 °C, respectively. PCR products were resolved on 2% agarose gels. The resultant PCR products were sized against a 1 kb DNA molecular ladder (Bioline, London, UK). The expected PCR product sizes ranged between 300-1200 bp. PCR products were puri ed using QIAquick PCR Puri cation Kit (Qiagen, Germantown, MD, USA) and commercially Sanger-sequenced (Inqaba Biotec, Muckleneuk, Pretoria, South Africa).