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Sample GSM5410612

Query DataSets for GSM5410612

Status

Public on Jul 01, 2021

Title

Sample type

SRA

Source name

Adult RiboTag x Adv-Cre mouse L4,5 DRG tissues

Organism

Mus musculus

Characteristics

genotype: RiboTag x Adv-Cre
developmental stage: Adult
injury: no injury
tissue: DRG

Treatment protocol

For lentiviral transduction, lentivirus was added to the culture at DIV 2. For re-plating assay using the embryonic DRG cultures, neurons were re-plated at DIV 5. Briefly, cultures were incubated in DMEM/0.05% Trypsin-EDTA mixture (1:1) for 5 min at 37°C, 5% CO2 and rinsed with culture medium described above. Cells were then dissociated by gentle pipetting and transferred to new culture plates coated with poly-D-lysine/laminin. Re-plated Neurons were incubated over night at 37°C with 5% CO2.

Growth protocol

Embryonic DRG cultures were generated as previously described. Embryonic DRG collected from embryonic day 13.5 mice were dissociated in 0.05% trypsin-EDTA and plated on poly-D-lysine/laminin-coated dishes in Neurobasal medium (Gibco) supplemented with 2% B-27 (Gibco), 1% Glutamax, 1 μM 5-fluoro-2'-deoxyuridine (Sigma), 1 μM uridine (Sigma), 1% penicillin-streptomycin, and 50 ng/ml 2.5S nerve growth factor (Envigo, BT-5017).

Extracted molecule

total RNA

Extraction protocol

RNA extraction and analysis procedures were performed with RNAqueous Total RNA isolation kit (Thermo, AM1931), RevertAid Reverse Transcriptase (Thermo, EP0441), and PowerUP SYBR green master mix (Thermo, A25918) from L4-5 DRG tissues or cultured embryonic neurons (E13, DIV 5), following the manufacturer’s instructions. The transcript levels were compared between conditions by the ΔΔCt method, using Gapdh transcript levels as an internal control.
RNA samples were prepared using RNAqueous Total RNA isolation kit (Thermo Fisher Scientific, AM1931). To analyze ribosome-associated RNA, Nanopore PCR-cDNA barcoding sequencing kit (Oxford Nanopore Technologies, SQK-PCB109) was used for library preparation. The quantified RNA samples (50ng) were subjected to reverse transcription reaction followed by strand-switching reaction as the manufacturer’s guide. The VN primer was annealed to the RNA to target poly A tail, and first-strand cDNA was synthesized by Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific, EP0752). The RNA-cDNA hybrid was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, A63880). The library from the Cre- negative control, Cre+ DRG without injury and Cre+ DRG with injury was subjected to barcoding process using barcode primers (Oxford Nanopore Technologies, BP01-BP12). PCR was performed using 2x LongAmp Taq Master Mix (New England Biolabs, M0287S) to select full-length transcripts followed by a second purification step using Agencourt beads (as described above). The barcoded libraries from the triplicates of the uninjured DRG tissue (labeled as u1, u2, u3) and from the triplicates of the injured DRG tissues (labeled as x1, x2, x3) were multiplexed with concentration normalization and loaded onto Flow Cell (Oxford Nanopore Technologies, FLO-MIN106D) as the manufacturer’s guide. The flow cells were used with more than 800 active pores from the initial pore check. MinION and MinKNOW (Oxford Nanopore Technologies, ver. 20.10.3) was used for sequencing with the integrated basecaller guppy for real-time fast-basecalling. Sequencing process was setup with duration of 72 hours, Q-score of 7, and fast-basecalling of Guppy (ver. 4.2.2). After basecalling and de-multiplexing from MinKNOW, the sequencing reads were subjected to NanoStat and NanoPlot to check quality of reads with scores and lengths 95, and mapped using Minimap2 (option, -ax splice:hq -uf) 96 with a reference Mus musculus (GRCm38.p6, GCA_000001635.8). Samtools were used for sorting and indexing the mapped reads 97 and edgeR was used for statistical analysis of differential levels between uninjured and injured DRG samples 36,37. To screen the target genes for downstream analysis, total 12,967 identified protein-coding genes were selected and plotted as x-y plot, with the x-axis of bulk-seq result indicating differential expression levels after sciatic nerve injury and the y-axis of the relative levels of ribosome-association efficiency after injury. To visualize the statistical significance from the edgeR analysis result, the relative level of -log10 P-value was indicated as the relative size of the circle. To analyze total DEGs of bulk-seq. from adult DRG tissues after sciatic nerve crush injury, three C57/BL6 mice were used as a set of a biological replicate. Right side sciatic nerves were crushed and L4,5 DRGs were dissected at 24 hours after injury. Left side L4,5 DRGs were subjected to uninjured DRG samples. Total RNA was extracted from DRG tissues using RNAqueous Total RNA isolation kit (Thermo Fisher Scientific, AM1931). Nanopore libraries were prepared from total RNA by using Nanopore Direct RNA sequencing Kit (Oxford Nanopore Technologies, SQK-RNA002) according to the manufacturer’s instructions. The RT adapter was annealed to the RNA for reverse transcription and reverse-transcribed RNA was then purified using Agencourt RNAClean XP magnetic beads (Beckman Coulter, A63880). The purified RNA was eluted by adding 21μl Elution Buffer and resuspending the beads, incubating at room temperature for 10 min, pelleting the beads again, and transferring the supernatant (pre-sequencing mix) to a new tube. Then the sequencing adapter was ligated to the library. Nanopore sequencing was performed as per manufacturer's guidelines using R9.4 SpotON flow cells (FLO-MIN106, ONT) with MinION sequencer winth MinKNOW software by setting duration 72 hours. Basecalled reads was mapped to the Mus musculus reference (GRCm38.p6, GCA_000001635.8) using MiniMap2 (option, -ax splice -uf -k14). Indexing and statistical analysis was done using Samtools and edgeR. Total three biological replicates were subjected to edgeR analysis for testing statistical significance of injury-responsive differential expression. To analyze CSDE1-associated RNA, mouse embryonic DRG spot-culture was used. Spot-culture DRG neurons were infected with control or 5’UTRm lentivirus at DIV2 and then lysed in TRAP lysis buffer at DIV5. The quantified total extracts were incubated with anti-CSDE1 antibody and the immunopricipitants were subjected to RNA extraction and library preparation same as described in RiboTag-seq. analysis. Two biological replicates were subjected to edgeR analysis to test statistical significance.
Nanopore direct RNA-Seq, PCR-cDNA seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Description

Control
Rpl22HA-immunoprecipitated RNA

Data processing

The flow cells were used with more than 800 active pores from the initial pore check. MinION and MinKNOW (Oxford Nanopore Technologies, ver. 20.10.3) was used for sequencing with the integrated basecaller guppy for real-time fast-basecalling. Sequencing process was setup with duration of 72 hours, Q-score of 7, and fast-basecalling of Guppy (ver. 4.2.2). After basecalling and de-multiplexing from MinKNOW, the sequencing reads were subjected to NanoStat and NanoPlot to check quality of reads with scores and lengths 95, and mapped using Minimap2 (option, -ax splice:hq -uf) 96 with a reference Mus musculus (GRCm38.p6, GCA_000001635.8). Samtools were used for sorting and indexing the mapped reads 97 and edgeR was used for statistical analysis of differential levels between uninjured and injured DRG samples 36,37. To screen the target genes for downstream analysis, total 12,967 identified protein-coding genes were selected and plotted as x-y plot, with the x-axis of bulk-seq result indicating differential expression levels after sciatic nerve injury and the y-axis of the relative levels of ribosome-association efficiency after injury. To visualize the statistical significance from the edgeR analysis result, the relative level of -log10 P-value was indicated as the relative size of the circle.
Nanopore sequencing was performed as per manufacturer's guidelines using R9.4 SpotON flow cells (FLO-MIN106, ONT) with MinION sequencer winth MinKNOW software by setting duration 72 hours. Basecalled reads was mapped to the Mus musculus reference (GRCm38.p6, GCA_000001635.8) using MiniMap2 (option, -ax splice -uf -k14). Indexing and statistical analysis was done using Samtools and edgeR. Total three biological replicates were subjected to edgeR analysis for testing statistical significance of injury-responsive differential expression. To analyze CSDE1-associated RNA, mouse embryonic DRG spot-culture was used. Spot-culture DRG neurons were infected with control or 5’UTRm lentivirus at DIV2 and then lysed in TRAP lysis buffer at DIV5. The quantified total extracts were incubated with anti-CSDE1 antibody and the immunopricipitants were subjected to RNA extraction and library preparation same as described in RiboTag-seq. analysis. Two biological replicates were subjected to edgeR analysis to test statistical significance.
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: Excel file, Differential expression list

Submission date

Jun 29, 2021

Last update date

Jul 01, 2021

Contact name

Yongcheol Cho

E-mail(s)

[email protected]

Phone

82-53-785-6190

Organization name

DGIST