title: GSM6675570: R90-2-4d; Stutzerimonas balearica; RNA-Seq
library_construction_protocol: RNA was extracted from the samples by column fecal RNAout Kit (TIANDZ). After the feces (0.5g) were ground to powder in liquid nitrogen, buffer A preheated by 1ml at 65 °C was added and mixed with 300ul buffer B and 200ul chloroform for 20 seconds. After swirling for 30 seconds, the supernatant was centrifuged, and then the buffer C of the same volume was mixed upside down and transferred to the adsorption column. After centrifugation, the penetrating solution was discarded and the 700ul universal column solution was washed once and then washed with 300ul universal column solution, and RNA was eluted with 100ulRNA elution solution. RNA is quantified using Qubit (Invitrogen, USA). RNA digested by DNase was removed rRNA by Ribo-Zero digestion Magnetic Kit (Gram-Negative Bacteria or Gram-positive Bacteria) (Epicentre, USA): adding Ribo-Zero Reaction Buffer and Ribo-Zero rRNA Removal Solution (Gram-Negative Bacteria or Gram-positive Bacteria), adding Ribo-Zero Reaction Buffer and Ribo-Zero rRNA Removal Solution (Gram-Negative Bacteria or Gram-positive Bacteria) to 40 μ l, reacting 10min at 68 ℃, then placing 5min at room temperature, adding the treated RNA to the pre-washed magnetic beads, mixing thoroughly immediately, placing 5min at room temperature, then reacting 5min at 50 ℃, immediately placing above 1min on the magnetic rack. Absorb the supernatant, add water to 180 μ l, add 3 M Sodium Acetate and Glycogen (10 mg/ml), add 600ul anhydrous ethyl alcohol, place at-20 ℃ for more than 1 h, centrifuge to get precipitation, add water to dissolve rRNA-depleted RNA. Take 10ng rRNA-depleted RNA from RNA library, construct library with KAPA Stranded mRNA-Seq Kit (KAPA Biosystems, USA): add KAPA Fragment,Prime and Elute Buffer (2X) to interrupt mRNA, react 6min at 94 °C, quickly put on ice; add 1stStrand synthesis Buffer and KAPA Script to synthesize the first chain of cDNA, reaction procedure: 25 °C 10 minutes, 42 °C 15 minutes, 70 °C 15 minutes; and then add 2ndStrand Marking Buffer (2x) and 2ndStrand Synthesis EnzymeMix for cDNA second chain synthesis, 16 °C reaction for 1 h. After purification with AMPure XP Beads (Agencourt, USA), add KAPA A-Tailing Buffer (10x) and A-Tailing Enzyme for terminal repair and add A, reaction procedure: 30 °C 30min, 65 °C 30 min; add Adaptor, KAPA Ligation Buffer (5x) and KAPA T4 DNA Ligase joint, 20 °C DNA Ligase reaction for 15 min; 200~300bp range of ligated cDNA was obtained by two-step AMPure XP Beads purification. PCR amplification was carried out by adding KAPA HiFi HotStart Ready Mix (2x) and KAPA Library Amplification Primer Mix (10x). The PCR reaction conditions were as follows: pre-denaturation at 98 °C for 45 Sec, denaturation at 98 °C for 15 Sec, annealing at 60 °C for 30 Sec, extension at 72 °C for 30 Sec;, and then purified with 1-fold volume of AMPure XP Beads to obtain a library for sequencing.