SRX18014672 GSM6675557_r1 SRP404411 PRJNA894131 SRS15522921 GSM6675557 GSM6675557 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014673 GSM6675558_r1 SRP404411 PRJNA894131 SRS15522922 GSM6675558 GSM6675558 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014674 GSM6675559_r1 SRP404411 PRJNA894131 SRS15522923 GSM6675559 GSM6675559 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014675 GSM6675560_r1 SRP404411 PRJNA894131 SRS15522924 GSM6675560 GSM6675560 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014676 GSM6675561_r1 SRP404411 PRJNA894131 SRS15522925 GSM6675561 GSM6675561 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014677 GSM6675562_r1 SRP404411 PRJNA894131 SRS15522926 GSM6675562 GSM6675562 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014678 GSM6675563_r1 SRP404411 PRJNA894131 SRS15522927 GSM6675563 GSM6675563 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014679 GSM6675564_r1 SRP404411 PRJNA894131 SRS15522928 GSM6675564 GSM6675564 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014680 GSM6675565_r1 SRP404411 PRJNA894131 SRS15522929 GSM6675565 GSM6675565 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014681 GSM6675566_r1 SRP404411 PRJNA894131 SRS15522931 GSM6675566 GSM6675566 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014682 GSM6675567_r1 SRP404411 PRJNA894131 SRS15522930 GSM6675567 GSM6675567 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014683 GSM6675568_r1 SRP404411 PRJNA894131 SRS15522933 GSM6675568 GSM6675568 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014684 GSM6675569_r1 SRP404411 PRJNA894131 SRS15522932 GSM6675569 GSM6675569 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014685 GSM6675570_r1 SRP404411 PRJNA894131 SRS15522935 GSM6675570 GSM6675570 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014686 GSM6675571_r1 SRP404411 PRJNA894131 SRS15522934 GSM6675571 GSM6675571 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014687 GSM6675572_r1 SRP404411 PRJNA894131 SRS15522936 GSM6675572 GSM6675572 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014688 GSM6675573_r1 SRP404411 PRJNA894131 SRS15522937 GSM6675573 GSM6675573 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014689 GSM6675574_r1 SRP404411 PRJNA894131 SRS15522939 GSM6675574 GSM6675574 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014690 GSM6675575_r1 SRP404411 PRJNA894131 SRS15522938 GSM6675575 GSM6675575 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014691 GSM6675576_r1 SRP404411 PRJNA894131 SRS15522940 GSM6675576 GSM6675576 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014692 GSM6675577_r1 SRP404411 PRJNA894131 SRS15522941 GSM6675577 GSM6675577 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014693 GSM6675578_r1 SRP404411 PRJNA894131 SRS15522943 GSM6675578 GSM6675578 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014694 GSM6675579_r1 SRP404411 PRJNA894131 SRS15522942 GSM6675579 GSM6675579 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014695 GSM6675580_r1 SRP404411 PRJNA894131 SRS15522944 GSM6675580 GSM6675580 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014696 GSM6675581_r1 SRP404411 PRJNA894131 SRS15522945 GSM6675581 GSM6675581 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014697 GSM6675582_r1 SRP404411 PRJNA894131 SRS15522947 GSM6675582 GSM6675582 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000 SRX18014698 GSM6675583_r1 SRP404411 PRJNA894131 SRS15522946 GSM6675583 GSM6675583 RNA-Seq TRANSCRIPTOMIC cDNA 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. Illumina NovaSeq 6000