Name: GSM6947264
Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: For E. coli and G. metallireducens monocultures, total RNA extractions were performed using Trizol according to the manufacturer's protocol. RNA was extracted from F. succinogenes strain UWB7 monoculture & co-culture pellets using the RNeasy Mini Kit following the protocol for purification of total RNA from plant cells and tissues and filamentous fungi, using liquid N2 method of cell lysis, QIAshredder homogenization, on-column DNase I digestion, and elution in 30 µL of RNAse-free water. (1) Overview: EMBR-seq libraries were prepared according to the protocol described in Wangsanuwat et al. 100 ng of total RNA were used as input material for all libraries. For the libraries denoted “EMBR-seq” and “EMBR-seq+”, blocking primers were added during the polyadenylation step and reverse transcription step. For the libraries denoted “No depletion” and “RNase H”, water was added instead of blocking primers during these two steps. In the libraries denoted “RNase H” and “EMBR-seq+”, the RNase H treatment was performed as described below. In the libraries denoted “No depletion” and “EMBR-seq”, the RNase H treatment was skipped; the amplified RNA (aRNA) product of in vitro transcription (IVT) was used directly for the library reverse transcription step as described in Wangsanuwat et al. (2) Polyadenylation: 2 μL of total RNA (50 ng/μL) was mixed with 1 μL of 5x first strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, comes with Superscript II reverse transcriptase, Invitrogen Cat. # 18064014], 1 uL of blocking primer mix, 0.1 μL 10 mM ATP, and 0.1 μL E. coli poly-A polymerase (New England Biolabs, Cat. # M0276S). The samples were incubated at 37°C for 10 mins. In the “No depletion” and “RNase H” libraries, 1 μL of nuclease-free water was added instead of the blocking primer mix. (3) Reverse transcription: The polyadenylation product was mixed with 0.5 μL 10 mM dNTPs (New England Biolabs, Cat. # N0447L), 1 μL reverse transcription primers (25 ng/μL, Supplementary Table 2), and 1.3 μL blocking primer mix, and heated to 65°C for 5 mins, 58°C for 1 min, and then quenched on ice. In the “No depletion” and “RNase H” libraries, 1.3 μL of nuclease-free water was added instead of the blocking primer mix. This product was then mixed with 1.2 μL 5x first strand buffer, 1 μL 0.1 M DTT, 0.5 μL RNaseOUT (Thermo Fisher Scientific, Cat. #10777019), and 0.5 μL Superscript II reverse transcriptase, and then incubated at 42°C for 1 hr. Immediately afterwards, the samples were heat-inactivated at 70°C for 10 mins. (4) Second strand synthesis: The reverse transcription product was mixed with 33.5 μL nuclease-free water, 12 μL 5x second strand buffer [100 mM Tris-HCl (pH 6.9), 23 mM MgCl2, 450 mM KCl, 0.75 mM β-NAD, 50 mM (NH4)2SO4, Invitrogen, Cat. # 10812014], 1.2 μL 10 mM dNTPs, 0.4 μL E. coli ligase (Invitrogen, Cat. # 18052019), 1.5 μL DNA polymerase I (Invitrogen, Cat. # 18010025), and 0.4 μL RNase H (Invitrogen, Cat. # 18021071), and incubated at 16°C for 2 hrs. The cDNA was purified with 1x AMPure XP DNA beads (Beckman Coulter, Cat. # A63881) and eluted in 24 μL nuclease-free water that was subsequently concentrated to 6.4 μL. (5) In vitro transcription: The product from the previous step was mixed with 9.6 μL of in vitro transcription mix (1.6 μL of each ribonucleotide, 1.6 μL 10x T7 reaction buffer, 1.6 μL T7 enzyme mix, MEGAscript T7 Transcription Kit, Thermo Fisher Scientific, Cat. # AMB13345) and incubated at 37°C for 13 hrs. The IVT product was mixed with 6 μL ExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher Scientific, Cat. # 78200.200.UL) and incubated at 37°C for 15 mins. Next, it was treated with 5.5 μL fragmentation buffer (200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc) at 94°C for 3 mins and immediately quenched with 2.75 μL stop buffer (0.5 M EDTA) on ice. The fragmented aRNA was size-selected with 0.8x AMPure RNA beads (RNAClean XP Kit, Beckman Coulter, Cat. # A63987) and eluted in 18 μL nuclease-free water. In the libraries denoted “No depletion” and “EMBR-seq”, the fragmented and cleaned-up in vitro transcription product was used directly for Illumina library preparation by reverse transcription and PCR, as described previously (18, 30). In the “RNase H” and “EMBR-seq+” libraries, the RNase H treatment was performed before proceeding to Illumina library preparation. (6) RNase H treatment for monocultures: After ExoSAP treatment, RNA fragmentation, and bead-based size selection, the aRNA concentration was measured on a NanoDrop One spectrophotometer. 1000 ng of aRNA was mixed with 500 ng (3.2 μL) of RNase H probe mix, 3.2 μL of RNase H buffer mix, and nuclease-free water to reach a total volume of 15 μL for each reaction. This mixture was preheated at 65°C for 5 mins and quenched on ice. 1 μL of RNase H (Invitrogen, Cat. # 18021071) was added and the reaction was incubated at 16°C for 30 mins. Finally, the RNase H product was size-selected with 1x AMPure RNA beads and eluted in 15 μL nuclease-free water, and then concentrated to 5 μL volume for Illumina library preparation. (7) RNase H reaction for co-cultures: For co-culture samples, the RNase H probe mix was altered to include probes for depleting fungal rRNA along with bacterial rRNA. 1000 ng of aRNA derived from co-culture samples was mixed with 850 ng (5.5 μL) of co-culture RNase H probe mix, which consisted of: 100 ng each of the two fungal 28S probes with 50 ng each of the two fungal 18S, fungal 5.8S, and ten bacterial RNase H probes. The aRNA and probes were mixed with 3.2 μL RNase H buffer mix and nuclease-free water to reach a total volume of 15 μL for each reaction. The reactions were preheated at 65°C for 5 mins and quenched on ice. 1 μL of RNase H was added and the reaction was incubated at 16°C for 30 mins. The RNase H product was size-selected with 1x AMPure RNA beads and eluted in 15 μL nuclease-free water, and then concentrated to 5 μL volume for Illumina library preparation. OTHER (EMBR-seq+). Method is detailed in Heom, Wangsanuwat et al.