Rneasy mini kit

HepG2 cells were cultured
in 48-well plates (5–10 × 10³ per well) in
a complete medium containing Dulbecco’s modified Eagle’s
medium (DMEM, Sigma-Aldrich, #D6429) supplemented with 10% heat-inactivated
fetal bovine serum (FBS, Gibco, #1050064) and penicillin (100 U/mL)/streptomycin
(100 μg/mL) (Gibco, #15140122) in a humidified atmosphere at
37 °C with 5% CO₂. At a confluency of ∼70%,
cells were treated with a medium containing 0.5 μM Bex, 12.5
μM Bex, or 0.5 μM XCT, 12.5 μM XCT or 0.5 μM
BRF110, 12.5 μM BRF110 for 24 h. Following treatment, total
RNA was isolated according to the RNeasy Mini Kit protocol (Qiagen,
Hilden, Germany) and cDNA was synthesized using the PrimeScript RT
reagent Kit (Takara, RR037A) using oligo-dT and random hexamers. Quantitative
PCR was performed in a 7500 Real-Time PCR System (Applied Biosystems,
Carlsbad, CA, USA) using 2× SYBR Select Master Mix (Applied Biosystems,
#4472918). Gene expression analysis was carried out using the 2^–ΔΔCT relative quantification
method. Triplicate reactions were performed for each sample, and the
average CT was calculated for the quantification analysis. GAPDH was
used as an endogenous reference control. Human SREBP-1c (F, 5′-CCATGGATTGCACTTTCGAA-3′;
R, 5′-GGCCAGGGAAGTCACTGTCTT-3′). Human GAPDH (F, 5′-TGGGCTACACTGAGCACCAG-3′;
R, 5′-GGGTGTCGCTGTTGAAGTCA-3′).

Embryos and explants of indicated stages were treated with RNAlater (Ambion) overnight at 4°C. Total RNA was isolated using RNeasy Mini Kit (Qiagen) and reverse-transcribed using Superscript III First-strand Synthesis System (Invitrogen). Real-time PCR was performed in Rotor-gene 6 instrument (Corbett Research) using QuantiTect SYBR Green PCR Kit (Qiagen).
A representative result of 3 separate experiments is shown. The following primers were used:Table

Gene	Direction	Sequence
Eomesodermin (Eomes)	Forward	5′-GCA GAG GTT CTA CCT ATC-3′
	Reverse	5′-CAT TGC TTG AGG TGC TAG G-3′
Goosecoid (Gsc)	Forward	5′-ACA ACT GGA AGC ACT GGA-3′
	Reverse	5′-TCT TAT TCC AGA GGA ACC-3′
EF1α	Forward	5′-CCT GAA CCA CCC AGG CCA GAT TGG TG-3′
	Reverse	5′-GAG GGT AGT CAG AGA AGC TCT CCA CG-3′
VegT	Forward	5′-CAA GTA AAT GTG AGA AAC CGT G-3′
	Reverse	5′-CAA ATACAC ACA CAT TTC CCG-3′
Xbra	Forward	5′-GGA TCG TTA TCA CCT CTG-3′
	Reverse	5′-GTG TAG TCT GTA GCA-3′
Xnr1	Forward	5′-AAC CAT CAC TTA TCA ATA GG-3′
	Reverse	5′-TGT AGG CCA GTA AAA TCA TTA AC-3′
XPofut1	Forward	5′-GAT GCC AGG CGG GTG TCT TGT TT-3′
	Reverse	5′-AGG CCT GAT TTC ATG GAG TCT TTA-3′
XVent1	Forward	5′-TTC CCT TCA GCA TGG TTC AAC-3′
	Reverse	5′-GCA TCT CCT TGG CAT ATT TGG-3′

Primary HFKs and HPV18-transfected isogenic lines (1 × 10⁵) were cultured on γ-irradiated J2 fibroblasts in epidermal growth factor containing E medium [25 (link)] in 10 cm dishes until 80% confluent. J2 fibroblasts were then removed and HFKs harvested by trypsinisation. Cells were pelleted, washed with PBS, snap frozen and stored at –80 °C. RNA was extracted using the RNeasy Mini Kit following the manufacturer’s protocol (Qiagen) and DNase I treated. Libraries were prepared using TruSeq Stranded mRNA Library Prep kit for NeoPrep (Illumina) using 100 ng total RNA. Libraries were pooled and run as 75 cycle pair end reads on a NextSeq 550 (Illumina) using a high output flow cell.
Sequencing reads were aligned to GRCh37 human genome merged with HPV18 genome (AY262282.1) using STAR aligner (v2.5.2b) [55 (link)]. Reads mapping to genes were counted by the same software. Normalisation of read counts and differential expression analysis between HFK and HPV18 samples was performed with DESeq2 (v.1.26.0) R Bioconductor package [56 (link)], taking into account the paired sample design of the experiment. Gene set enrichment analysis was done using GAGE (v.2.36.0) R Bioconductor package [57 (link)] with MSigDB gene set database. The computations were performed on the CaStLeS infrastructure (http://doi.org/10.5281/zenodo.3250616) at the University of Birmingham.
The PlotPCA function in DESeq2 was used to calculate the principal components of rlog transformed RNA-Seq expression data of the 500 most variable genes. The ClusterSignificance [58 (link)] R Bioconductor package was used to evaluate statistical significance of group separation compared to permuted data. The P-value was calculated using 10⁴ permutations.

RNA was extracted from 1 × 10⁶Smchd1+/+;EμMycTg/+ and Smchd1MD1/MD1;EμMycTg/+ lymphoma cells using Qiagen RNeasy Minikit as per the manufacturers instructions. Libraries were prepared using Illumina’s TruSeq RNA sample preparation kit as per the manufacturers instructions and submitted to the Australian Genome Research Facility for quality control, library preparation and sequencing on the Illumina HiSeq 2000 platform using 100 base, paired end or single-end reads. Base calling and quality scoring were performed using Real-Time Analysis (version 1.17.21.3) and FASTQ file generation and de-multiplexing using CASAVA (version 1.8.2). Reads from FASTQ files were aligned to the mouse genome (mm10) using Subread (version 1.10.5) (26 (link)) and summarized at the gene-level using the featureCounts procedure (27 (link)). Subsequent analysis was carried out using the ‘edgeR’ (28 (link)) and ‘limma’ (14 ) Bioconductor software. The counts were transformed into CPM to standardize for differences in library-size and filtering was carried out to retain genes with a baseline expression level of at least 0.5 CPM in three or more samples. Data were TMM normalized (3 (link)) and an MDS plot was generated (Figure 1B) before linear models using various weighting strategies (described below) were fitted to summarize over replicate samples. Moderated t-statistics were used to assess differential expression between Smchd1MD1/MD1 and Smchd1+/+ (wild-type) samples, with genes ranked according to their FDR (22 ). These data are available under GEO series accession number GSE64099.
Smchd1 has been shown to have a role in the regulation of clustered protocadherins and imprinted genes in diverse tissues including whole embryo, adult brain, embryonic fibroblasts, placenta and malignant and normal B cells (30 (link)–32 (link)). We obtained gene sets for these two classes of genes to use as true positives (TPs) in our analysis. To identify protocadherins, we used regular expression matching to look for this term in the gene name field of the annotation of the filtered data set, which returned eight genes (out of a total of 71 in the mouse genome). A comprehensive set of imprinted mouse genes was downloaded from http://www.mousebook.org/imprinting-gene-list and matched to the expressed genes in this data set using Gene Symbols. In total, 46 genes out of the 150 in the original list were matched.

Quantitative PCR (qPCR) was performed to characterize the relative Wolbachia infection level in the S2 cell lines and flies. The protocol was similar to prior qPCR amplification using the single-copy wsp and su(fk)C genes of bacterial and host origin, respectively [65 (link)]. S2 cells were quantified using a hemocytometer to obtain 10⁶ cells. The S2 cells or DSR females were homogenized in 100 μl STE with 0.4 mg/ml proteinase K to extract DNA as previously described [66 (link)].
For qRT-PCR, RNA extractions were performed on groups of 10 ovaries or 10 testes dissected from one-day post eclosion infected and uninfected Drosophila adults using the RNeasy Mini Kit (Qiagen). DNA contamination was removed with RNase-Free DNase Set (Qiagen). RNA quality and quantity was checked with NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Inc.). Synthesis of cDNA was performed with Superscript II Reverse Transcriptase (Invitrogen) using specific primer for Ance (AnceQ F 5'-CGGTCACGTTCGATTGGTTG-3' and AnceQ R 5'-CTTCGGTTTCCACGTTGGTTC-3') and Actin gene (ActinQ F 5'-GCTGACCGTATGCAAAAGG-3' and ActinQ R 5'-GCTTGGAGATCCACATCTG-3'). Primers were designed based upon D. simulans genbank sequences for Ance and Actin (genbank accession number: NM_057696 and NM_079486, respectively]. qRT-PCR was performed separately with the AnceQ F/R and ActinQ F/R primer pairs using a Miniopticon system (BioRad) with a Platinum SYBR Green qPCR superMix (Invitrogen). qRT-PCR reactions were conducted using a 2 minute step at 50°C, 2 minute step at 95°C and 40 cycles of 15 seconds at 95°C and 30 seconds at 56°C. A fluorescence measurement was made at the end of each cycle. A melting curve analysis was performed at the end of the amplification program to examine for primer-dimers or nonspecific amplification. Assays were performed on two (D. simulans and D. melanogaster wild type) or three (D. melanogaster Ance mutants) independent experiment replicates for each sex and infection type. As an examination for variability, duplicate qRT-PCR reactions were performed for each set of ovaries or testes with both the Ance and Actin primers. Relative expression of Ance gene was calibrated against Actin using the ΔΔC_T calculation method [67 (link)] with:

Expression variation=2−ΔΔCT
For comparisons of males and females, the above was modified as follows: