Truseq rna samples prep kit

cDNA libraries were prepared from high quality RNA using an Illumina TruSeq RNA sample prep kit following the manufacturer’s instructions (Illumina, San Diego, CA, USA). For each sample, 3 μg of total RNA was used for cDNA preparation. Briefly, mRNA was purified from total RNA and then fragmented. First strand cDNA synthesis was performed using SuperScript II Reverse Transcriptase (Applied Biosystems Ltd.) subsequently synthesising the second strand using components of the Illumina TruSeq RNA samples prep kit. Adaptors were ligated to the cDNA which was then enriched by PCR. Final individual cDNA libraries were validated on the Agilent Bioanalyser 2100 using the DNA 1000 Nano Lab Chip kit, ensuring that library fragment size was ~260 bp and library concentration was >30 ng/μl. After quality control procedures, individual RNAseq libraries were pooled based on their respective sample-specific-6 bp adaptors and sequenced at 100 bp/sequence single-end reads using an Illumina HiSeq 2000 sequencer. Approximately 16 million sequences per sample (Mean ± SD = 15,964,874 ± 1,903,207) were generated.

Pre-B cells were isolated from the bone marrow of recipient mice that had been transplanted 3 weeks earlier with sgTfap4/Eµ-MYC/Cas9 or sgControl/Eµ-MYC/Cas9 FLCs. B cells were enriched from bone marrow of all long bones by staining with a cocktail of biotinylated antibodies against TER119 (Ly76), MAC-1 (M1/70), GR-1 (RB6-8C5) for 20 min on ice, washed, then incubated with MagniSort Streptavidin Negative Selection Beads (Thermo Fisher Scientific) according to the manufacturer’s protocol to remove undesired erythroid and myeloid cells. The supernatant was transferred into a fresh tube, washed, and resuspended in 100 µl of a cocktail of fluorochrome-conjugated antibodies against B220 (RA3-6B2), IgM (5.1), c-KIT (2B8) and 1 × 10⁶ live (PI negative) pre-leukaemic pre-B cells (B220⁺ sIgM⁻ c-KIT⁻) were FACS sorted, centrifuged, and resuspended in QIAzol Lysis Reagent (Qiagen) and stored at −80 °C. Total RNA was extracted using the QIAgen miRNeasy Mini Kit according to the manufacturer’s instructions with optional on-column DNAase digestion. An input of 100 ng of total RNA was used to prepare mRNA libraries and indexed using the TruSeq RNA samples Prep Kit (illumina) according to the manufacturer’s protocol. The samples were sequenced on an Illumina NextSeq using paired-end sequencing. RNA-sequencing analysis are detailed in the Supplementary Material.