GSM9065894: Liver metastases from control (shSCR) 4T1 cells, replicate 2; Mus musculus; RNA-Seq
Following mice euthanasia, livers were extracted, and liver metastases from mice injected with 4T1 shSCR (n = 3) and 4T1 shZdhhc17 (n = 3) were dissected under the microscope. RNA was then isolated from the dissected metastases using Trizol reagent (Thermo Scientific). RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-8000 (Nanodrop Technologies) and RNA integrity and concentration were assessed using the Bionalyzer RNA Nano kit (Agilent). Per sample, 500 ng of total RNA were used as input for RNA-sequencing library preparation. Using the Illumina Stranded mRNA Sample Prep Kit (protocol version: # 1000000124518 v02 (April 2021)), poly-A containing mRNA molecules were purified from the total RNA input using poly-dT oligo-attached magnetic beads. The purified mRNA was then fragmented, converted into first strand cDNA in a reverse transcription reaction using random primers and Actinomycin D, and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using dUTP to achieve strand specificity. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which pre-index anchors were ligated preparing the fragments for dual indexing. Anchor-ligated fragments were then purified using magnetic beads. Finally, enrichment PCR was carried out to enrich those DNA fragments that have anchor-ligated DNA fragments and to add indexes and primer sequences for cluster generation.
GEO
GSM9065894_r1