Picopure rna isolation kit

Directly prior to laser dissection microscopy (LDM), 5 µm-thick sections of testis tissue were mounted on Superfrost glass microscope slides (Thermo Fisher Scientific) and stained with Hematoxylin and Eosin as described previously (Jan et al., 2017 (link)). For each patient, 500 histologically pachytene-like spermatocytes were individually laser dissected and pooled as described previously (Jan et al., 2017 (link)). Laser-dissected cells were captured in silicon-coated adhesive caps (Adhesive cap 500 opaque tube, Zeiss) and were lysed at 42°C in 10 µl of extraction buffer provided in the PicoPure RNA isolation kit (Arcturus). Cell lysates were stored at −80°C until further use. All procedures were performed under RNase-free conditions. Total RNA was isolated from cell lysates using the PicoPure RNA isolation kit (Arcturus) according to the manufacturer's protocol, including an on-column DNase treatment. RNA was eluted in 10 µl elution buffer. Subsequently, the RNA was concentrated to a volume of 5 µl with a speed vacuum centrifuge for 8 min. Total RNA isolated from 500 pooled cells was SPIA-amplified using the Ovation RNAseq V2 System (Nugen) as described previously (Jan et al., 2017 (link)).

Cells were prepared for FACS sorting as described above and sorted into RNA Extraction buffer. RNA was extracted with PicoPure^TM RNA Isolation Kit (Arcturus) according to manufacturer´s protocol. The RNAseq analysis and read mapping was performed as a service at EMBL. HT-seq python package (version, 0.5.3p9, with flags–mode = union–stranded = no–type = exon) was used to count the number of reads aligned to the reference genes (genome build GRCm38.p4, Ensembl). Data were uploaded to Gene Expression Omnibus (GEO, accession no. GSE90160). Differential gene expression analysis was done with R using package EdgeR (v3.2.4) and a gene was considered differentially expressed if the adjusted p-value was <0.05. Gene ontology analysis was performed using PANTHER gene analysis tool (pantherdb.org). RNA analysis data are available in Supplementary Table S3.

The scFv library was generated as described before [25 (link)] starting from total RNA obtained from a pool of B cells purified from the CSF of two relapsing remitting (RR) MS patients both positive for the presence of oligoclonal bands and without treatments: a female, age 40 (disease onset at 36), EDSS 3 and a male, age 29 (disease onset 27), EDSS 3.5. Briefly, total RNA was extracted from 2 × 10⁴ B cells using PicoPure^TM RNA Isolation Kit (ARCTURUS) and First strand cDNA was synthesized using random hexamers and SuperScript^TM III RT (Invitrogen) following the manufacturer’s protocol. Each family of VH and VL genes were amplified separately from the first strand cDNA by PCR using 1 unit of recombinant ExTaq Polymerase (TaKaRa) and specific VH primers or VL primers [26 (link)]. For the VH chains, the 3' primer was specific for the IgG subclass. Single VH and VL genes were combined to obtain two equimolar VH and VL mixtures, which were amplified and assembled as described by Sblattero et al [26 (link)]. The assembled PCR products coding for the scFv were then ligated into the phagemid vector pDAN5 and transformed into DH5αF' E. coli cells by electroporation. Positive cells were selected on ampicillin agarose plate. Single clones obtained from the transformation were analysed by PCR, enzymatic fingerprinting (BstOI) and sequencing to evaluate the library diversity.