|
| Status |
Public on May 25, 2012 |
| Title |
Tagmented single-cell level cDNA with Tn5 transposase #3 |
| Sample type |
SRA |
| |
|
| Source name |
E14tg2a ES cell line, tagmentation
|
| Organism |
Mus musculus |
| Characteristics |
cell line: E14tg2a
cell type: embryonic stem cell
library preparation method: tagmentation with hyperactive Tn5 transposase
|
| Growth protocol |
Cells were grown in GMEM/10% FBS of ES cell grade supplemented with NEAA, Glutamax, Pyruvate, beta-mercaptoethanol, and LIF.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Single-cell cDNA preparation: Total RNA was prepared from E14tg2a ES cells with the QIAGEN RNeasy Mini Kit. 10 pg of total RNA equivalent to the single cell level was used for 1st strand cDNA synthesis with Oligo (dT) tagged by V1 primer. After the addition of poly (A) tail to the 3'-end of the 1st strand, the 2nd strand was synthesized with Oligo (dT) tagged by V3 primer. The single-cell level cDNA was amplified by 20 cycles of PCR with V1 and V3 primers.
Library preparation with Sonication: A single cell cDNA was fragmented by a Covaris E210 AFA Ultrasonicator. Libraries were prepared with the Illumina mRNA-seq kit (RS-100-0801). The obtained product (single cell cDNA) was subjected to fragmentation by a sonicator, end-repair, adaptor ligation, and library preparation with PCR.
Library preparation with hyperactive Tn5 transposase: The libraries were prepared with the Nextera DNA preparation kit (Illumina compatible) (Epicentre, an Illumina company) with some modification. 100 ng of single cell cDNA was suspended in HMW buffer, and tagmentation (fragmentation and tagging with the adaptors) was performed with the Nextera enzyme (hyperactive Tn5 transposase), incubating at 55C for 5 min. Tagged and fragmented cDNA was purified with DNA Clean & Concentrator-5 (Zymo Research), and then the libraries were prepared by PCR with Nextera PCR enzyme, Nextera primer cocktail, and Nextera adaptor 2 according to the following program: one cycle of 72C for 3 min and 95 C for 30 s (one cycle), 13 cycles of 95C for 10s, 62C for 30s, 72C for 3min.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
ES_SC_Nextera_3
Technical replicate 3 of 3.
Single cell lysate (total RNA).
|
| Data processing |
Illumina Pipeline RTA 1.8.70.0 / BCL Converter 1.7.1 / CASAVA 1.7.0 was used for image analysis and base calling.
Reads were aligned to the mouse mm9 genome and RefSeq transcripts using Partek Flow. Up to 2 mismatches in 28 bases in the seed was allowed.
Aligned reads were mapped to RefSeq gene definitions and annotated by Partek Genomics Suite.
RPKMs were calculated by Partek Genomics Suite.
Reads mapped to multiple sites were allocated according to the relative distribution of reads mapped to unique locations.
Genome_build: mm9
|
| |
|
| Submission date |
May 23, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kenta Yashiro |
| E-mail(s) |
k.yashiro@qmul.ac.uk
|
| Phone |
+44 (0)20 7882 8235
|
| Organization name |
Barts and The London School of Medicine and Dentistry, Queen Mary University of London
|
| Department |
Willaim Harvey research Institute
|
| Lab |
Translational Medicine and Therapeutics
|
| Street address |
Charterhouse Square
|
| City |
London |
| ZIP/Postal code |
EC1M 6BQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE38198 |
Tn5 transposase-mediated library preparation of single-cell cDNAs for RNA-seq |
|
| Relations |
| SRA |
SRX149541 |
| BioSample |
SAMN00998419 |
