E gel ex

HEK293T cells cultured in 6-well plates were washed with PBS and harvested by adding 600 μl TRIzol (Life Technologies cat#15596026) directly to the cells. Total RNA was extracted with Direct-zol RNA Miniprep Plus kit (Zymo Research cat# R2070) following manufacturer’s protocol. RNA integrity was confirmed by the presence of 18 S and 28 S bands on a 2% E-Gel EX (Invitrogen cat# G402002). RNA libraries were prepared with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB cat# E7490L) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB cat# E7760L). 500 ng RNA was used at input, and the quality of final libraries were confirmed by qPCR and TapeStation. Sequencing of the libraries was performed by the Biopolymers Facility at Harvard Medical School using NovaSeq6000.

All samples were analyzed for the presence of HPV by PCR, using the MGP primer set as previously described by Söderlund-Strand et al. [13 (link)]. The five forward and the five reverse MGP primers amplify 158–168 nt of the L1 gene depending on the HPV type. In the MGP PCR protocol for Luminex^®, the AmpliTaq Gold polymerase (Life technologies/Applied Biosystems, CA, USA) was used. A modified MGP PCR protocol using the proofreading Phusion Hot Start II High-fidelity DNA polymerase (Thermo Fisher Scientific, MA, USA) was established for NGS. Here, the modified MGP PCR mastermix consisted of: 1 × Phusion HF buffer, 0.2 mM dNTPs, 1 μM primer mix, 0.2 U Phusion DNA polymerase. Sample input was 5 μl to a total reaction volume of 20 μl with a final concentration of 0.1 μM of each primer. The modified MGP PCR protocol was run with the following cycling conditions: 98°C 30 s, 5 cycles 98°C 5 s, 42°C 5 s and 72°C 5 s, followed by 45 cycles 98°C 5 s, 64°C 5 s, 72°C 5 s prior to cooling to 4°C. All samples were stored at -20°C until further processing and analysis. To evaluate the outcome of each PCR, 5 μl of all PCR products were analyzed on a 4% E-gel EX (Life technologies/Invitrogen, CA, USA).

PCR products were deemed worthy of sequencing when producing a strong, clear band at the correct product size when visualised using an Invitrogen E-Gel iBase Real-Time Transilluminator with 2% SYBR safe E-Gel EX agarose gels run for 10 mins. Products were sent to Source BioScience (Nottingham, UK) for cleanup prior to forward and reverse sanger sequencing. The MLST primers used were gene-specific and in the case of MLST genes just the M13 primers (M13_adaptor_F: 5’-TGTAAAACGACGGCCAGT-3’ and M13_adaptor_R: 5’-CAGGAAACAGCTATGACC-3’) were used if these adaptors were included in the initial PCR to generate the product. MEGAX
^{47 (link)}
was used for all analysis of sequences with manual checking of both forward and reverse chromatograms. Editing of sequences included trimming and then alignment to produce consensus sequences was undertaken using ClustalW. Nucleotide BLAST (NCBI) database queries and searches against the
Wolbachia MLST database were combined to determine if new alleles and strain types were present in our collection. We also submitted our sequences to GenBank and obtained accession numbers.

Total RNA from spleens and lymph nodes was extracted (RNeasy Maxi Kit, Qiagen) from each unimmunized heavy chain and kappa chain only transgenic rat (OmniRat, Open Monoclonal Technology Inc., Palo Alto, CA, USA) and antibody sequences were amplified as previously described^{13 (link)} except for different primers used during reverse transcription (Table S2). We chose to interrogate the antibody repertoires found in secondary lymphoid organs as opposed to peripheral blood due to the higher number of B cells. Correct PCR product sizes were verified on an agarose gel (E-Gel EX; Invitrogen) and quantified with fluorometry (Qubit; Life Technologies), pooled at approximately equimolar concentrations and each sample pool was re-quantified before sequencing on an Illumina MiSeq (MiSeq Reagent Kit v3, 600-cycle). All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee of Scripps Research and approved by the Institutional Research Boards of Scripps Research.

Multiple taxonomic gene regions for all studied lichens were selected and amplified based on the latest phylogenetic-based studies available for each lichen genus as outlined in detail below. All PCR reactions were carried out using the Platinum Direct PCR Universal Master Mix - Kit (Thermo Fisher Scientific Inc) in a Mini Amp Plus Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific, Waltham, USA).
All PCR products were checked by gel electrophoresis using 1% E-gel EX (Invitrogen, Thermo Fisher, Waltham, USA) in an E-Gel Power Snap instrument (Invitrogen, Thermo Fisher, Waltham, USA).
Successful PCR products were purified using the NucleoSpin Gel and PCR Clean-up Kit (Marchery Nagel, New England, Canada) according to the manufacturer’s standard protocol. Subsequently, they were sent for Sanger sequencing carried out by Azenta (Göttingen, Germany) with the primers that were for the amplification of the respective gene regions. All generated DNA sequences were deposited at the National Center for Biotechnology Information (NCBI) GenBank.