GSM9040079: p53null SOFT mammary tumors Sample 6; Mus musculus; RNA-Seq
Tumor tissues were mechanically digested in DNA lysis buffer (4 M guanidine thiocynate, 100 mM Tris/HCl pH 7.4, 30 mM EDTA pH 8.0, 1% Triton X-100, 100 mM b-mercaptoethanol). Lysates were incubated with acid-washed diatomaceous earth to enhance nucleic acid binding, followed by silica column-based purification (Promega). Column-bound DNA was purified through sequential washes with guanidine- and ethanol-based DNA wash buffers. RNA was isolated from the DNA flowthrough by sodium acetate-phenol:chloroform extraction. The aqueous phase was incubated with acid-washed diatomaceous earth and ethanol to promote RNA binding to the column, followed by purification through guanidine- and ethanol-based RNA wash buffers. DNA was used in WES, and RNA was used in RNA-Seq and RT-qPCR analyses. RNA-Seq libraries were prepared as previously described (60), with the following modifications. Briefly, 1 mg total RNA was enriched for poly(A) transcripts using oligo(dT)-conjugated magnetic beads (KAPA mRNA HyperPrep Kit, Roche, Cat # 08098123702), followed by fragmentation, template-switch reverse transcription, and dual-index barcode PCR amplification (Roche, Cat #KK8725). To minimize ribosomal RNA contamination, two consecutive rounds of bead binding using 25 mL were performed, and intact poly(A) RNA was eluted in 5 mL nuclease-free water at 65 °C for 2 mins. Custom-magnetic immobilizers—3 mm × 1 mm neodymium disc magnets (K&J Magnetics) embedded at a 45 o angle in polydimethylsiloxane (PDMS) at the base of 0.2 mL PCR tubes—were used to localize magnetic beads during washes and minimize carryover. Enriched RNA was chemically fragmented and reverse-transcribed using a modified SMART-Seq3 protocol (61). Briefly, RNA was fragmented by heating a 1:1 mixture of RNA and 2X fragmentation buffer (50 mM Tris-HCl pH 8.3, 60 mM NaCl, 6 mM MgCl₂, 13.3% PEG-8000, 0.27% Triton X-100, and 2.7 µM oligo(dT)₃₀) at 94 °C for 2 min, to generate a target library size of 300–400 bp (corresponding to ~120–220 bp inserts). Samples were cooled on ice and combined with 3 µL of 4X reverse transcription master mix (25 mM Tris-HCl pH 8.3, 30 mM NaCl, 3 mM MgCl₂, 4 mM neutralized GTP, 32 mM DTT, 2 mM each dNTP, 4 µM template-switching oligo), 1 U/µL Ribolock RNase inhibitor (Thermo, Cat #EO0381), and 2 U/µL Maxima H Minus Reverse Transcriptase (Thermo, Cat #EP0751). First-strand synthesis was performed at 42 °C for 90 mins, followed by 10 cycles of 2 mins each at 50 °C and 42 °C, with enzyme inactivation at 85 °C for 5 mins. cDNA fragments were size-selected on a 2% agarose gel (200-650 bp), excised, dissolved in solubilization buffer, incubated with acid-washed diatomaceous earth, followed by column-based purification (Promega) and eluted with pre-heated elution buffer. Libraries were amplified using KAPA HiFi master mix and dual-index barcodes, for 16–18 cycles of 98 °C 15 sec, 60 °C 30 sec, 72 °C 1 min, with amplification monitored from cycle 14 by gel electrophoresis. Final libraries were ethanol-precipitated with linear acrylamide and sodium acetate and stored at –80 °C. Single-end, 150-bp sequencing was performed on a HiSeq 4000 (The University of California, San Francisco, Center for Advanced Technology).