Maxwell rsc simplyrna cells kit

RNA was extracted using the Maxwell® RSC simplyRNA Cells Kit (Promega, Madison, Wisconsin, USA) following the manufacturer’s instruction. The library preparation was carried out according to the TruSeq RNA Exome protocol (Illumina, San Diego, CA, USA) as previously described [28 (link)]. The experiment was repeated in triplicate.

Total RNA was extracted by using a Maxwell RSC simplyRNA Cells Kit (Promega, Fitchburg, WI, USA) and Maxwell RSC Instrument in accordance with the manufacturer's protocol. Total RNA (500 ng) was reverse transcribed to cDNA by using a PrimeScript 1st strand cDNA synthesis kit (TaKaRa Bio Inc., Shiga, Japan) in accordance with the manufacturer's protocol. SINEUP-GFP RNA, EGFP RNA and GAPDH RNA expression were quantified by polymerase chain reaction (PCR) amplification using SYBR Premix Ex Taq (Tli RNaseH Plus) (TaKaRa Bio Inc.). Relative expression was analyzed by using the 2^−ΔΔCT method (31 (link)).
The following qRT-PCR primers were used:

MCF-10A cells were plated in 6 cm tissue culture dishes in triplicate using complete DMEM/F12 and cultured overnight. The media was then exchanged for fresh MCBD-170 media [20 (link)] and cultured for 24 h. Cells were harvested with trypsin followed by soybean trypsin inhibitor (Thermo Fisher Scientific, Asheville, NC, USA) and RNA isolated using the Maxwell RSC simplyRNA cells kit (Promega, Madison, WI, USA) as per the manufacturer’s instructions. DNA was isolated in parallel using Ultra-Pure phenol:chloroform:isoamyl alcohol (Thermo Fisher Scientific). Nucleic acid purity and integrity was confirmed by agarose gel and sent to Genewiz (South Plainfield, NJ, USA) for RNAseq and WGS. Raw and processed data are available online (Gene Expression Omnibus accession no. GSE128388).

Total RNA was extracted from cells using the Maxwell^® RSC simplyRNA Cells Kit (Promega). Genomic DNA was digested with DNase, according to the manufacturer’s instructions. Extracted RNA was retrotranscribed to cDNA, and qRT-PCR was performed using TaqMan^TM Universal PCR Master Mix and the following TaqMan^TM probes spanning at exon junctions (Applied Biosystems): human ATF4 (Hs00909569_g1), human DDIT3/CHOP (Hs99999172_m1), human GAPDH (Hs03929097_g1). For analysis of XBP1 splicing, qRT-PCR was performed using SYBR™ Green PCR Master Mix and the specific primers for the spliced human XBP1 (forward 5′-CTG​AGT​CCG​CAG​CAG​GTG​CA-3′, reverse 5′-GGT​CCA​AGT​TGT​CCA​GAA​TGC​CCA​A-3′), as well as for human GAPDH (forward 5′-CAA​ATT​CCA​TGG​CAC​CGT​CA-3′, reverse 5′-GAC​TCC​ACG​ACG​TAC​TCA​GC-3′). Values were normalized to GADPH (ATF4 and CHOP) or HPRT1 (XBP1s) levels. Relative expression levels were determined using the 2^−ΔΔCt method.

K562 cells (5 × 10⁶ cells/mouse) were injected subcutaneously into the flank of NOD.Cg-Prkdc scidIl2rgtm1Wjl/SzJ (NSG) mice (strain #005557). After 8 days, mice bearing tumours were divided into three groups of five, matched by age and sex: (1) control group received PBS (100 μL/injection); (2) the second group received intra-tumoural injections of Ce-49 sh loaded GenVoy LNPs (Precision Nanosystems) (0.3 mg/kg injection/mouse in 100 μL/injection); (3) the third group received intra-tumoural injections of 5-Aza (Sigma) (1 mg/kg injection/mouse in 100 μL/injection). Treatments were performed daily for 10 days. Tumour size was determined by callipers according to the formula: tumour volume (mm3) = = L × W × H, where L is length, W is width and H is height.
Mice were maintained at standard temperature (20–23 °C) with 30–70% humidity and a 12 light/12 dark cycle. At the end of the treatments, the mice were sacrificed, tumours were recovered and lysed for RNA/DNA extraction. Maxwell RSC tissue DNA kit (Promega, AS1610) and Maxwell RSC simply RNA cells kit (Promega, AS1390) were used. Genomic DNAs samples were analyzed by Diagenode EPIC methylation array after bisulfite conversion. RNA samples were subjected to RNA sequencing. Study was performed within the approved IACUC protocol 10016 of City of Hope.

Established/PDC cells were cultivated for 48 h in the respective media. At confluence of 70–80%, cells were placed on ice and washed twice with ice-cold PBS, mechanically scratched from the plate in 1 ml of ice-cold PBS, and centrifuged at 4 °C/400 g for 5 min. Pelleted cells were stored in − 80 °C until all cells were collected for RNA isolation. RNA was isolated using the Maxwell RSC simplyRNA Cells Kit (no. AS1390, Promega, Germany). Cell RNA isolation kit was used according to the manufacturer’s instructions. Total RNA was stored at − 80 °C until further processing and gene expression analysis. For PDX samples, RNA was isolated from fresh-frozen PDX tumor tissue using the PARIS (Ambion) isolation kit.