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Sample GSM5735911

Query DataSets for GSM5735911

Status

Public on Dec 15, 2021

Title

4cell embryo - WAGO1_rep1

Sample type

SRA

Source name

4 cells embryos

Organism

Ascaris suum

Characteristics

tissue: 4 cells embryos
5'-end modification: mono- and triphosphate
argonaute ip antibody: AsWAGO1
treatment: RNA 5' Pyrophosphohydrolase

Treatment protocol

Growth protocol

Ascaris 4-cell embryos were developed in incubator at 30 C shaking at 100 rpm for 64hr.

Extracted molecule

total RNA

Extraction protocol

Male and female germline tissues were extracted from worms and washed in PBS, frozen whole or following dissection in liquid nitrogen, and stored at -80°C. Argonaute IPs were carried out using affinity purified rabbit antibodies pre-bound to Protein A Dynabeads (Fisher Scientific) (5-10 ug of antibody per 100 ul of beads). Germline or embryo whole cell lysates were mixed with Protein A Dynabeads and rotated overnight at 4°C. Protein A Dynabeads were recovered and washed with high-salt buffer (50 mM Tris–HCl, pH 7.4, 1M NaCl, 1mM EDTA, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate) three times. The beads were resuspended in 250 ul of Proteinase K buffer containing 200 µg/ml Proteinase K and incubated for 1 hr at 37°C. RNA was extracted using Trizol LS (Invitrogen) adapted for small RNA recovery by precipitation using 2 volumes of isopropanol, 15 µg of GlycoBlue (Invitrogen), and 30 minutes incubation at -80°C followed by centrifugation at 18,500 x g for 35 minutes at 4°C. The RNA was treated with RppH (25 units, New England Biolabs) in Thermopol buffer for 5’ independent cloning. Both untreated and RppH-treated samples were done for AsALG-1 and AsALG-4 samples. Following RppH treatment, samples were repurified with Trizol LS (Invitrogen) (adopted for small RNAs extraction) and stored at -80°C. RNA was isolated using Trizol or LS Trizol (Invitrogen). Total RNA samples were fractionated into small RNA (< 200 nt) and long RNA (>200nt) using the Monarch RNA cleaner (New England Biolabs).
Small RNA libraries were prepared using the Small RNA-Seq Library Prep Kit (Lexogen). Both 5’-phosphate and 5’-phosphate independent libraries (RppH-treated) were prepared. Libraries from 4-cell embryos were also prepared using 18-30 nt gel purified RNA using the SMARTer smRNA-Seq Kit (Clontech) and NEXTflex-Small-RNA-Seq (New England Biolabs) with similar results. RNA >200 nt was treated with TURBO DNase (Ambion) and rRNA depletion carried out using RiboCop rRNA Depletion Kit for Human/Mouse/Rat (Lexogen). Long RNA (>200 nt) libraries were made using CORALL Total RNA-Seq Library Prep Kit (Lexogen). Both small and long RNA libraries were sequenced 150 nt from both ends using the Illumina NovaSeq 6000 System.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Description

WAGO1_4cell_rep1
small RNA
Library made with NEB kit
WAGO1_64hr.nr.fa.gz

Data processing

Adapters were clipped and artifacts removed with FASTX Toolkit 0.0.13 (fastx_clipper and fastx_artifacts_filter).
18-30nt were filtered.
Annotated against local databases of rRNA, tRNA, mtRNA, miRNAs, repeats and transcripts with bowtie with following parameters: -f -v 1 --best.
Reads were mapped to Ascaris genome with bowtie2 with following parameters: -f -x
Genome_build: WGS Project JACCHR01 (assembly ASM1343314v1)
Supplementary_files_format_and_content: Nonredundant reads (nr) of processed fasta files: ids >1_23421, where 1 is order number of the read and 23421 is number of reads.

Submission date

Dec 14, 2021

Last update date

Dec 19, 2021

Contact name

Maxim Vladimirovich Zagoskin

E-mail(s)

maxim.zagoskin@utk.edu

Organization name

The University of Tennessee, Knoxville

Department

Biochemistry & Cellular and Molecular Biology