GSM7730222: RNAseq data from intratumoral CD45+ cells from B20-4.1.1 treated MMTV-PyMT breast tumors sample 3; Mus musculus; RNA-Seq
Tumors were harvested, minced and dissociated with Tumor Dissociation Kit enzymes (Miltenyibiotec, #130-096-730) and incubated in gentleMACS C tubes (Miltenyibiotec, #130-093-237) on gentleMACS™ Octo Dissociator (Miltenyibiotec, #130-095-937) using 37C_m_TDK_1 programme. Then, 10% FBS DMEM medium were added to stop enzyme reaction and cell suspension was passed through a 40µm strainer. Next, centrifugation was performed at 300g, 5min, 4ºC and cell pellet was resuspended in RBC 1X Lysis buffer (eBioscience, #00-4333-57). After washing with 1x PBS, cell suspension was resuspended in Flow cytometry staining (FACS) buffer (PBS, 1% Bovine Serum Albumin, 1% FBS, 0.01% Sodium Azide) and used for cell staining and FACS sorting. CD45+ cells were sorted using BD Influx directly into RLT Lysis buffer. Then, RNA was extracted using RNAeasy Mini Plus Kit (Qiagen, #74134) and quantified in LabChip GX Touch Nucleic Acid Analyzer (PerkinElmer). RNA quality was determined by Agilent's 2100 Bioanalyzer Lab-Chip technology. Only samples with RNA Integrity Number (RIN)>7 was used for sequencing. Variable amounts of total RNA samples, with RNA Quality score of 7.0 on average (PerkinElmer LabChip analyzer), were analysed. They were processed into cDNA sequencing libraries with the "QuantSeq 3' mRNA-Seq Library Prep Kit (FWD) for Illumina" (Lexogen, Cat.No. 015) with a UMI Second Strand Synthesis module. Briefly, library generation is initiated by reverse transcription with oligodT priming, followed by a random-primed second strand synthesis. Random primers feature a 6 nt long Unique Molecular Identifier 5' tag. Primers from both steps contain, in addition, Illumina-compatible sequences. Libraries are completed by PCR, and sequenced on an Illumina instrument (see below) by following manufacturer's protocols.{This library preparation kit generates directional libraries stranded in the sense orientation (the read1 (the only read in single read format) has the sense orientation (--library-type fr-secondstrand in TopHat, --stranded=yes in HTSeq)}