GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5769597

Query DataSets for GSM5769597

Status

Public on Oct 14, 2022

Title

Human prostate (1D cDNA)

Sample type

SRA

Source name

Human prostate tissue

Organism

Homo sapiens

Characteristics

tissue: Human prostate
nanopore library type: 1D cDNA

Extracted molecule

polyA RNA

Extraction protocol

Poly(A)+ RNA (5 μg) from human prostate were purchased from Clontech
50 ng of poly(A)+ RNA from human prostate tissue were used as templates for cDNA synthesis following the SMART-seq2 protocol with some modifications. The reverse transcription and template-switching reaction was performed by Maxima H minus reverse transcriptase (Thermo Scientific, #EP0751) under the following conditions: 42°C for 90 min, 85°C for 5 min. PCR amplification of first-strand cDNA using KAPA HiFi ReadyMix (KAPA Biosystems, #KK2602) was performed by incubating at 95°C for 3 min, followed by 13 cycles of (98°C for 20 s, 67°C for 20 s, 72°C for 4min) with a final extension at 72°C for 5 min. PCR products were treated with Exonuclease I (NEB, #M0293) to remove unused primers and then purified using 0.8X volume of SPRIselect beads (Beckman Coulter, #B23318). 1D Nanopore libraries were then constructed using 1 μg of amplified cDNA according to the standard SQK-LSK109 protocol. cDNA products were end-repaired and dA-tailed using NEBNext Ultra II End Repair/dA-Tailing Module (NEB, # E7546) by incubating at 20°C for 20 minutes and 65°C for 20 min. End-prepared cDNA were purified with 1X volume of AMPure XP beads and eluted in 60 μl of Nuclease-free water. Adapter ligation was then performed using NEBNext Quick T4 DNA ligase (NEB, #E6056) at room temperature for 10 min. After ligation, the libraries were purified using 0.45X volume of AMPure XP beads and short fragment buffer (SFB) to enrich all fragments equally.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Description

processed data file:
30ClontechTissue_1D_cDNA_N2_R0_abundance.esp
30ClontechTissue_1D_cDNA_N2_R0_updated.gtf

Data processing

Basecalling was performed in fast mode with Guppy (version 3.6.0) for all Nanopore sequencing data in this study (https://community.nanoporetech.com/downloads). Basecalling of direct RNA and cDNA libraries were done using config files rna_r9.4.1_70bps_fast.cfg and dna_r9.4.1_450bps_fast.cfg respectively.
Consensus sequences from multiple copies of cDNA found in each long read of an LRCA library were obtained by running SPIRIT (https://github.com/Xinglab/SPIRIT). In brief, nhmmer is first used to find splint sequences from each read with the parameter ‘-T 8 --max’. Sequences with similar lengths separated by each pair of neighboring splint sequences were considered as copies of cDNA. If three or more copies of cDNA were identified, their consensus was obtained using abPOA (https://github.com/Xinglab/abPOA).
Reads were then mapped to either the SIRVome or hg19 reference genome using minimap2 (v2.17) with parameters “-ax splice --splice-flank=no -w 4 -k 14” and either SIRV transcript annotations or GENCODE v34 annotations (https://www.gencodegenes.org/human/release_34lift37.html).
Alignment files (SAM format) were next processed by ESPRESSO (https://github.com/Xinglab/espresso) using default settings and either SIRV transcript annotations or GENCODE v34 annotations. Reads that either map to the mitochondrial genome or contain large continuous insertions (≥20 nt) in the raw alignment were filtered out by ESPRESSO.
Genome_build: hg19, SIRVome
Supplementary_files_format_and_content: .esp files are tab-delimited matrices generated by ESPRESSO that contain read count estimates for all identified isoforms (rows) across all samples (columns); .gtf files generated by ESPRESSO are in GTF format and provide genomic coordinates for all identified transcript isoforms and their corresponding exons

Submission date

Jan 03, 2022

Last update date

Oct 14, 2022

Contact name

Yi Xing

E-mail(s)

[email protected]

Organization name

Children's Hospital of Philadelphia

Department

Department of Pathology and Laboratory Medicine