Rna storage buffer

Total RNA extraction from PBMCs and the HCC827 cell line was carried out in a laminar flow hood under RNAse-free conditions using Trizol (Thermo Fisher Scientific, Fremont, CA, USA). The isolated RNA was dissolved in RNA storage buffer (Ambion, Austin, TX, USA) and stored at −80 °C until used. Amplification of the β-actin, as a house-keeping gene, was performed to verify the RNA integrity, as it has been previously described [25 (link),26 (link)].

RNAseq libraries enriched for low-abundance transcripts were prepared for AML cell lines, lymphomas, and FACS-sorted PreB and ProB cells as follows. RNA was extracted using Trizol reagent (Thermo Fisher Scientific), DNase-treated, then polyadenylated transcripts were isolated using the Dynabead mRNA purification kit (Thermo Fisher Scientific). Purified mRNA was fragmented by heating to 98°C for 30 min in RNA storage buffer (Ambion), then converted to cDNA with random primers using the Superscript III RT kit (Invitrogen). Second-strand synthesis was performed in the presence of dUTP, then Illumina adapters were ligated onto the dsDNA fragments. To preserve strand identity, the uracil-containing cDNA strand was digested using USER enzyme (NEB), then cDNA was amplified using adapter-specific PCR primers and purified using Agencourt AMPure XP beads (Beckman Coulter). The samples were subsequently enriched for low-abundance transcripts by Duplex-Specific Nuclease (DSN) treatment (Illumina) followed by low-cycle PCR, then gel-purified and cleaned up using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Libraries were sequenced by paired-end 76 bp on an Illumina HiSeq2500.

RNA was isolated from spleen tissue using Trizol reagent (Invitrogen, Carlsbad, CA) and then precipitated with isopropanol and dissolved in RNA storage buffer (Ambion, Austin, TX). First strand cDNA was synthesized by ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo, Osaka, Japan) following manufacturers’ instruction. Specific primers for each gene were designed using primer3 software. Q-PCR was performed with primers as shown in Table 1.
Amplification of 1ul of cDNA was performed by SYBR mixture using iQ^TMSupermix PCR Master Mix (Bio-Rad Laboratories, Hercules, CA, USA). The PCR program used an initial step of 95^°C for 3 min for pre-denaturation, followed by denaturation at 95^°C, annealing and extension at 58°C using the CFX connector (Bio-Rad Laboratories, Hercules, CA, USA). Quantization of relative gene expression was calculated by the comparative Ct method (2^−ΔΔCt) as described by the manufacturer. Data were normalized to GAPDH mRNA levels. Three independent experiments were carried out to study mRNA levels.