Monarch total rna extraction kit

Total RNA was extracted from 10⁷ hybridoma cells using the Monarch^™ total RNA extraction kit (New England BioLabs, Ipswich, MA, USA) following manufacturer’s instructions. Two μg of RNA served as template for cDNA synthesis using oligo dT and murine leukemia reverse transcriptase as implemented in the OneTaq RT PCR kit (New England Biolabs). A panel of oligonucleotides designed to amplify mouse Ig V genes as described by Wang et al (81 (link)) was used to PCR amplify and then sequence V_H and V_L from each hybridoma. The encoded protein sequences are shown in Supplementary Figure 5.

Total RNA was extracted from the patient FNA samples, the resulting BL cell lines, and NSG-BL mouse tumors using the Monarch Total RNA extraction kit (New England Biolabs). Starting with 1–5 µg total RNA, we enriched mRNA molecules using oligo-dT magnetic beads to capture polyA-tailed mRNA molecules. We then proceeded to prepare strand-specific RNA-seq libraries following the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Final library concentration and fragment size were confirmed with a Qubit 4 fluorometer (Thermo Fisher Scientific) and fragment analyzer (Agilent), respectively. These libraries were sequenced with paired-end reads (2 × 75 bp) on the NextSeq 550 system (Illumina, Inc.). Raw sequencing data files from this study are deposited in the NCBI’s database of Genotypes and Phenotypes (dbGaP) with accession number phs001282.v2.p1.

Total RNA from intestinal tissue and organoids at day 4 of culture was purified using the Monarch total RNA extraction kit, according to the manufacturer's protocol (New England Biolabs, USA). Concentration of RNA in samples was determined using a spectrophotometer DS-11Fx (DeNovix, USA). For reverse transcription, 0.5 μg of total RNA was transcribed using the M-MLV Reverse Transcriptase, according to the protocol (Bio-Rad: Cat.# 1708841). Quantitative RT–PCR was performed on a thermocycler QuantStudioTM 3 (Thermofisher Scientific, USA), using FastStart Universal SYBR Green master (Roche), 200 nM of each specific primer (excepting primers for Chromogranin A, which were used at 60 nM), and 1μL of cDNA. The following cycling parameters were used for all the markers: 1 cycle of 95 °C for 4 min; 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 30 s; 1 cycle of 72 °C for 5 min; melting curve analysis over a temperature range of 95 to 60 °C. Primers were synthesized by IDT (sequences are listed in Supplementary Table S2). Actin was used as a reference gene. Relative gene expression levels were determined using the standard curve method. Standard-curve points and unknown samples were performed in technical triplicates and replicates, respectively, and at least two biological replicates were analyzed per experimental condition.

The carborundum abrasion method was used to isolate the epidermis-enriched leaf RNA as described^{20 (link)} with modifications. C. roseus cv. Little Delicata leaves of approx. two centimeters in length (1 g) were collected in a 50 ml conical tube, then 1 g of carborundum (silicon carbide, 320 grit, Thermo Scientific, Waltham, USA) was added to the tube. After adding 3 ml RNA protection buffer from the Monarch® Total RNA extraction kit (New England Biolabs, Ipswich, USA), the mixture was vortexed for 30 s to release epidermis RNA. The remaining RNA isolation was performed according to the manufacture’s protocol. The qRT-PCR primers are listed in Supplementary Table 3.