title: GSM6704010: 4T1 cells, untreated, rep1; Mus musculus; RNA-Seq
library_construction_protocol: 10-50 mg of freshly collected cell pellets are lysed with 700 ml of Qiazol reagent.  After addition of chloroform, the homogenate is then separated into aqueous and organic phases by centrifugation. RNA partitions to the upper, aqueous phase, while DNA partitions to the interphase and proteins to the lower, organic phase or the interphase.  The upper, aqueous phase is extracted, and ethanol is added to provide appropriate binding conditions for all RNA molecules from 18 nucleotides upwards. The sample is then applied to the miRNeasy Mini spin column, where the total RNA binds to the membrane and phenol and other contaminants are efficiently washed away.  On-column DNAse digestion is also performed to remove any residual genomic DNA contamination followed by additional washes.  High quality RNA is then eluted in 60 ml of RNase-free water. Sequencing libraries were prepared with the RNA HyperPrep Kit with RiboErase (HMR) kit (Roche Sequencing Solutions) from 500ng total RNA.  Following the manufacturer's instructions, the first step depletes rRNA from total RNA.  The remaining RNA is DNase-digested to remove any genomic DNA contamination. Samples are then purified, fragmented, and primed for cDNA synthesis.  Fragmented RNA is then reverse transcribed into first strand cDNA using random primers.  The next step removes the RNA template and synthesizes a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA.  Pure Beads (KAPA Biosystems) are used to separate the ds cDNA from the second strand reaction mix resulting in blunt-ended cDNA.  A single 'A' nucleotide is then added to the 3' ends of the blunt fragments.  Multiple indexing adapters, containing a single 'T' nucleotide on the 3' end of the adapter, are ligated to the ends of the double-dtranded cDNA, preparing them for hybridization onto a flow cell.   Adapter-ligated libraries are amplified by PCR, purified using Pure Beads, and validated for appropriate size on a 4200 TapeStation using D1000 Screentape (Agilent Technologies, Inc.). The DNA libraries are quantitated using the KAPA Biosystems qPCR kit (Sigma) and are pooled together in an equimolar fashion, following experimental design criteria. NovaSeq Standard- Each pool is denatured and diluted to 350pM with 1% PhiX control library (Illumina) added.  The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge (Illumina), as determined by the number of sequencing cycles desired, and sequenced on a NovaSeq6000 instrument (Illumina) following the manufacturer's recommended protocol.