title: 16s rDNA of mus musculus:acute liver failure with NAFLD-FMT 7
design: The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R(5'-GGACTACHVGGGTWTCTAAT-3') [1] by T100 Thermal Cycler PCR thermocycler (BIO-RAD, USA). The PCR reaction mixture including 4 L 5  Fast Pfu buffer, 2 L 2.5 mM dNTPs, 0.8 L each primer (5 M), 0.4 L Fast Pfu polymerase, 10 ng of template DNA, and ddH2O to a final volume of 20 L. PCR amplification cycling conditions were as follows: initial denaturation at 95  for 3 min, followed by 27 cycles of denaturing at 95  for 30 s, annealing at 55  for 30 s and extension at 72 for 45 s, and single extension at 72  for 10 min, and end at 4 . The PCR product was extracted from 2% agarose gel and purified using the PCR Clean-Up Kit (YuHua, Shanghai, China) according to manufacturers instructions and quantified using Qubit 4.0 (Thermo Fisher Scientific, USA). Purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina Nextseq2000 platform (Illumina, San Diego,USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).