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| Status |
Public on Sep 29, 2023 |
| Title |
Fs+Ar RNase H Rep1 |
| Sample type |
SRA |
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| Source name |
strain UWB7, A. robustus
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| Organisms |
Fibrobacter succinogenes; Anaeromyces robustus |
| Characteristics |
cell line: strain UWB7, A. robustus
input material_for_library_preparation: total RNA (RNeasy), followed by poly-A depletion
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| Extracted molecule |
total RNA |
| Extraction 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.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
RNase H
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| Data processing |
In the sequencing libraries, read 1 contains the sample barcode. Read 2 is mapped to the bacterial or fungal transcriptome. Prior to mapping, read 1 was trimmed to 25 bases, read 2 was trimmed to 51 bases, and only reads containing valid sample barcodes on read 1 were retained. Next, the reads were mapped to the appropriate reference transcriptome using Burrows-Wheeler Aligner (BWA) version 0.7.15 with default parameters.
Assembly: Escherichia coli K12 substr. MG1655 (ASM584v2); Geobacter metallireducens GS-15 (ASM1292v1); Fibrobacter succinogenes strain UWB7 (GCA_900142945.1); Anaeromyces robustus (Anaeromyces robustus v1.0); Caecomyces churrovis (Caecomyces churrovis A v1.0)
Supplementary files format and content: Text file where column 1 contains gene names or gene IDs and column 2 contains gene counts.
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| Submission date |
Jan 20, 2023 |
| Last update date |
Sep 29, 2023 |
| Contact name |
Kellie Heom |
| Organization name |
University of California Santa Barbara
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| Department |
Chemical Engineering
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| Street address |
UC Santa Barbara
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| City |
Santa Barbara |
| State/province |
California |
| ZIP/Postal code |
93106 |
| Country |
USA |
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| Platform ID |
GPL33047 |
| Series (1) |
| GSE223404 |
Bacterial mRNA sequencing through targeted rRNA depletion for efficient RNA-seq in varied species and co-cultures |
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| Relations |
| BioSample |
SAMN32819520 |
| SRA |
SRX19124238 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6947250_rRNA_cds_4413-READ_COUNTS_c-bc.txt.gz |
12.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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