Sybr green master mix

For qRT-PCR on IL-7-cultured B cell precursors, cells were typically harvested on day 7 after transduction, dead/apoptotic cell-depleted, and snap-frozen on dry ice. For thymus and BM, cells were isolated freshly from 53BP1^−/− mice and snap-frozen on dry ice. Stimulated B cells were obtained from the spleen and cultured as described previously (Feldhahn et al., 2012 (link)). RNA was isolated as for RNA-seq, and cDNA was generated using the RevertAid cDNA synthesis kit (Thermo Fisher Scientific). qPCR was performed using a SYBR green master mix (Sigma JumpStart) on a StepOnePlus platform (Thermo Fisher Scientific). Information of primers, PCR conditions, and analysis can be found in the Supplemental Experimental Procedures. Analysis of the IGH locus for genomic loss was done as by Jankovic et al. (2013) (link) using the primers and conditions described.

siRNA targeting ESR1 (siESR1:SASI_Hs01_00078593; siESR1’:SASI_Hs01_00078594; siESR1”:SASI_Hs01_00078595), KMT2A (SASI_Hs01_00090459), KMT2B (siKMT2B:SASI_Hs01_00240396; siKMT2B’:SASI_Hs01_00240397; siKMT2B”:SASI_Hs01_00240398), KMT2C (SASI_Hs01_00037084), KMT2DE (SASI_Hs01_00145443) and KMT2E (SASI_Hs01_00238555) were obtained from MISSION RNA (Sigma-Aldrich, St. Louis, MO). Cells were transfected with siRNA and Lipofectamine^® RNAiMAX (Invitrogen) for 72 h according to the manufacturer's instructions. MISSION^® siRNA Universal Negative Control (Sigma-Aldrich) was used as knockdown control.
Total RNA was isolated from cells transfected with siRNA as indicated. Quantitative PCR (qPCR) was performed using SYBR Green dye on a Roche Applied Science LightCycler^® 2.0 Real-Time PCR System. Briefly, total RNA was reverse transcribed into cDNA using SuperScript III (Invitrogen, Carlsbad, CA) in the presence of random hexamers (Invitrogen). All reactions were performed in triplicates with SYBR green master mix (Sigma) and 20M of both the forward and reverse primers according to the manufacturer's recommended thermocycling conditions. Then the PCR products were subjected to melting curve analysis. Finally, the relative gene expression ratio of target genes to 18S rRNA for each sample was calculated. The primer sequences are listed in S1 Table.

Kyolic (Mission Viejo, CA, USA) provided the aged garlic extract. MTCC 1423 Lactobacillus rhamnosus was acquired from MTCC in India. Sigma-Aldrich (St. Louis, MO, USA) provided the iso-butyl methylxanthine and Tri reagent (Sigma-Aldrich, CA, USA). Gibco provided the fetal bovine serum (FBS) (Sigma-Aldrich, CA, USA). Applied Biosystems delivered the kits for cDNA synthesis and the SYBR green master mix (Sigma-Aldrich, CA, USA). Sigma provided commercially produced primer sequences. HiMedia Laboratories provided Molecular Biology grade ethanol with a purity of at least 99.8% and other chemicals (Mumbai, Maharashtra, India).

For gene expression analysis, total RNA was extracted using RNeasy Micro Kit (QIAGEN, MD) according to the manufacturer’s protocol. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific). Real-time PCR analysis was performed using the SYBR Green Master Mix (Sigma-Aldrich) following the manufacturer’s protocol on a 7500 Real-Time PCR system (Applied Biosystem). The relative expression of target genes was calculated using the delta–delta Ct method and normalized to the β-actin mRNA content. The Primer sequences are showed in Supplementary Tables 1 and 2.

Embryos were dechorionated in bleach and early embryos (pre-nc10)/eggs were selected in Voltalef medium. Pools of 15-20 embryos of each genotype were transferred to eppendorf tubes and dissociated in TRI Reagent (Sigma) with a plastic pestle. mRNA was extracted by adding chloroform, followed by 10 min centrifugation at 4°C and precipitation with isopropanol overnight. DNA was then pelleted by 10 min centrifugation at 4°C, washed in 70% ethanol, dried and resuspended in DEPC-treated water. Approximately 2 mg of RNA from each sample were DNAse treated with the DNA-free DNA Removal Kit (Invitrogen) in the presence of RiboLock RNase Inhibitor (Thermo Scientific). 1 mg of DNA-free RNA was then used for reverse transcription using M-MLV Reverse Transcriptase (Promega) in the presence of RiboLock. Samples were diluted 1:2 for RT-qPCR using SYBR Green Mastermix (Sigma) and primers detailed in Table S3.