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Sample GSM1582563

Query DataSets for GSM1582563

Status

Public on May 27, 2016

Title

miRNA-Seq analysis of breast variant human mammary epithelial cell from RM071 (m08323)

Sample type

SRA

Source name

m08323-1

Organism

Homo sapiens

Characteristics

submitted sample id: JOC207-RNA
donor_id: RM071
Sex: female
body site: Breast
histological type: Variant human mammary epithelial cell culture
is tumor: No
biomaterial_type: primary cell culture
cell type: breast variant human mammary epithelial cell

Extracted molecule

total RNA

Extraction protocol

library construction protocol: Refer to document 'miRNA3 - Plate Format miRNA Library Construction' from BC at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Description

design description: miRNA-Seq analysis of breast variant human mammary epithelial cell from RM071 (m08323) using Illumina HiSeq 2000
library name: m08323
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec. Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 25 uM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually: 12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot. Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 8.2
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: 2.108 ug
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN =>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70C for 2min then snap chilled on ice for 1min. Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added. Ligation incubated at 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70C for 2min then snap chilled on ice. 2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed. Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65C for 10 mins then quenched on ice. The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT. Reaction was heated at 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
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For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
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Data processing

Various levels of processed data files will be made available as this project proceeds.

Submission date

Jan 12, 2015

Last update date

May 15, 2019

Contact name

UCSF-UBC CENTER

Organization name

UCSF-UBC

Street address

UCSF-UBC