small RNA-seq of homo sapiens: adult female breast
Tissue biopsies (~100mg) were obtained at the time of primary treatment (mastectomy surgery), before any other cancer treatments (such as chemotherapy or radiotherapy) and flash frozen in liquid N2 prior to RNA extraction. Isolation of total RNA including the miRNA fraction was carried out using 1ml TRI Reagent (Thermo Fisher, USA) supplemented with the addition of 10mM MgCl2 to aid recovery of microRNAs.  Samples were then completely homogenised using a bead mill (Retsch Technology GmbH, Haan, Germany). Phase separation was carried out using chloroform.  Total RNA was precipitated from the aqueous phase by means of an overnight incubation at -20C with isopropanol.  RNA pellets were then ethanol-washed twice and re-suspended in RNase-free dH2O. RNA quality and concentration was assessed by Nanodrop spectrophotometry (Wilmington USA). QC and quantity measurement of the RNA was performed on an Agilent 2100 Bioanalyzer. All RNA samples had OD260/280 1.8 and RNA integrity number (RIN) 7. Selection of small RNAs for library preparation was performed using gel electrophoresis. Small RNA libraries were prepared using the NEB Next Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs Cat. NEB-E7580S). The libraries were sequenced with one lane on an Illumina NextSeq 500 instrument with 50-bp single-end reads at the Genomics Center of the Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel. Adaptor-only reads and low-quality reads were filtered out in the Illumina BaseSpace environment.