|
| Status |
Public on Feb 01, 2018 |
| Title |
MDA-MB-231_HM.LNm5_4 |
| Sample type |
SRA |
| |
|
| Source name |
mouse xenograft primary breast tumour
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: MDA-MB-231_HM.LNm5
tissue: mouse xenograft primary breast tumour
|
| Growth protocol |
Viable tumor cells resuspended in PBS were mixed with 10-30% Cultrex and inoculated (1x106 cells) into the right-side inguinal mammary gland of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immuno-deficient mice and then surgically resected at a size of 200-400mm3.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Resected primary tumors were disaggregated by digestion with collagenase I and bovine pancreatic DNase I at 37oC and filtered through a series of sieves prior to sorting for viable tumor cells by flow cytometry. Hydroxystilbamidine was used to exclude non-viable cells. Total RNA was prepared from sorted cells using the Trizol and the Direct-zol RNA mini kit with on-column DNase I digestion.
Library construction was by the PAT-seq approach. Breifly this involves a modified the ePAT approach to tagging adenylated RNA to generate libraries suitable for deep sequencing. Adenylated RNA is sequence specifically extended by dNTPs using Klenow polymerase and an annealed DNA anchor oligonucleotide. This takes advantage of the native function of DNA polymerase to extend an RNA primer from a DNA template in second strand synthesis. Importantly, any unwanted priming to internal poly(A)-tracts in RNA is avoided by a requirement for 3’ extension in subsequent fragment selection and reverse transcription. No ribosome depletion is necessary. Here, the anchor sequence was compatible with the Illumina index primers and included a 5’ biotin moiety to facilitate handling. In a second step, the 3’ tagged RNA was subject to limited fragmentation by RNase T1. This cleaves RNA after G-residues and ensures that cleavage is only possible within the body of the RNA, not the poly(A)-tract or the DNA sequence of the extended tag. The fragmented RNA was 5’ phosphorylated to allow RNA Ligase 2 mediated ligation of an Illumina compatible splinted-linker to the RNA fragments. Reverse transcription was primed from the anchor sequence. Note: All manipulations after limited fragmentation were performed in association with streptavidin magnetic beads. The cDNA PAT-seq libraries was eluted from beads, size-selected by Urea PAGE and amplified with primers that introduce the features for directional Illumina sequencing and indexing. Note: all reads run in 5’ to 3’ direction from unique sequence into a variable length of poly(A) homopolymers. This means that color balance is preserved and that any low fidelity within the homopolymers is limited to the end of the read.
Cluster Generation batch 1: 6pM of libraries per lane using Illumina c-bot. Illumina protocol 15006165 Rev J, July 2012.
Sequencing chemistry batch 1: 1 x 150bp sequencing using Illumina protocol 15035788 Rev A, Oct 2012
Cluster Generation batch 2: 9pM of libraries per lane using Illumina c-bot. Illumina Protocol 15035788 RevD, Apr 2014.
Sequencing chemistry batch 2: 150bp SR sequencing using Illumina HiSeq Rapid Mode and HiSeqV2 chemistry. Illumina Protocol 15035788 Rev D, Apr 2014
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Description |
Batch 2
Illumina index 16
HM_old_1
|
| Data processing |
In order to assign reads to H.Sapiens: Reads are first stripped of poly(A) sequence and adaptors using Tail Tools (https://github.com/Victorian-Bioinformatics-Consortium/tail-tools). Reads are then aligned to the human genome using bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml).
Reads assigned to the genome are then processed using the Tail Tools pipeline (https://github.com/Victorian-Bioinformatics-Consortium/tail-tools): Reads are aligned using bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Multi-mapping reads are assigned to one location at random. Alignments are extended where the stripped poly(A) sequence matches As present in the genome. A poly(A) tail is called present if there are at least four untemplated As remaining in the stripped sequence. Reads aligning to genes are counted. As 3'UTR annotations are not always accurate, reads up to 400 bases down-strand of a gene, but not extending into another gene on the same strand, are counted towards that gene. Total reads per gene, reads with a poly(A) tail per gene, and average poly(A) tail length are tabulated.
Genome_build: H. sapiens from release 82 of Ensembl
Supplementary_files_format_and_content: Names in the files are Ensembl gene ids. The ..._count.csv files give read counts for each gene in each sample. ..._tail.csv files give average poly(A) tail length for each gene in each sample. ..._tail_count.csv give the number of reads having a poly(A) tail. (The average poly(A) tail length is a less accurate estimate of tail length where there are fewer reads with a poly(A) tail.)
|
| |
|
| Submission date |
Jul 21, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew Pattison |
| E-mail(s) |
andrew.pattison@monash.edu
|
| Organization name |
Monash University
|
| Street address |
Wellington Rd
|
| City |
Melbourne |
| State/province |
Vic |
| ZIP/Postal code |
3800 |
| Country |
Australia |
| |
|
| Platform ID |
GPL18460 |
| Series (2) |
| GSE101745 |
Functional and genomic characterization of a xenograft model system for the study of metastasis in triple-negative breast cancer. |
| GSE111706 |
Metastasis in triple-negative breast cancer |
|
| Relations |
| BioSample |
SAMN07375445 |
| SRA |
SRX3026870 |
