Genomiphi hy kit

Sodium bisulfite modificationof denatured genomic DNA was performed as previously reported [82 (link)]. One microgram of genomic DNA per sample was converted using the EZ DNA methylation kit (Zymo Research Corp, Irvine, US-CA). Leukocyte DNA from healthy women and WGA (illustra ready-to-go GenomiPhi HY kit, GE healthcare, Little Chalfont, UK), were used as negative controls for methylation, whereas in vitro methylated leukocyte DNA, produced using M. SssI methyltransferase (New England Biolabs, Ipswitch, US-MA), served as a positive control.

The repertoires observed were supported using PCRs specific to three given alleles among five patients (Supplementary Table 3, Supplementary Fig. 2). Three alleles were selected that were present in several patients and absent in others. Primers were designed within these alleles to amplify each of them specifically (Supplementary Table 3). The specificity of the primer pairs were assessed by blasting them against the 1007 different msg-I alleles identified in the genomic plus expressed repertoires observed in the present work, as well as against the whole nucleotide collection (nr/nt). The PCRs were performed with the following parameters: 3 min at 94 °C followed by 35 cycles consisting of 15 s at 94 °C, 30 s at 56 °C, and 2 min at 72 °C, followed by a final extension of 7 min at 72 °C. The presence or absence in patients observed by PacBio CCS was confirmed by the specific PCRs (Supplementary Fig. 2). The sequences of the amplicons obtained using the Sanger technology showed 100% identity with those obtained using CCS. The positive DNA control was produced by random amplification of 2.5 µl DNA from the BAL of patient LA2 using the GenomiPhi HY kit (GE Healthcare), followed by a purification step using the columns of the QIAamp DNA Blood Mini Kit (Qiagen). It was also used for the set up of the PCR reactions.

Single cells of the protist Pseudotrichonympha sp. were collected from P. japonicus guts using a TransferMan NK2 micromanipulator (Eppendorf, Hamburg, Germany). Each protist cell was disrupted by adding 1% Tween 20 (Nacalai Tesque, Kyoto, Japan) (34 (link)), and bacterial cells that leaked out were collected using the micromanipulator and subjected to isothermal WGA by a GenomiPhi HY kit (GE Healthcare, Chicago, IL, USA), as described previously (11 (link)). All steps were conducted in a clean-room.