A-192

Since each single-cell library received a unique barcode, libraries can be pooled (multiplexed) and sequenced together. Per 96-well plate, the full volume (6 μL) of the single nuclei and negative controls were pooled together with 1 μL of the ten nuclei controls. Size selection was performed on a 2 % E-gel EX (Invitrogen) to isolate the mononucleosome fragments of approximately 280 bp (range of 200–400 bp). The DNA was eluted from the gel slices using Zymoclean gel DNA recovery kit (Zymo) according to the manufacturer’s protocol. The DNA quantity and quality were assessed using Qubit fluorometer (Invitrogen) and Bioanalyzer with High sensitivity chips (Agilent), respectively. For sequencing, clusters were generated on the cBot and single-end 50 nt reads were generated using the HiSeq2500 sequencing platform (Illumina, San Diego, CA, USA). In all runs, a pool of 192 libraries was sequenced on one lane of a flow cell.

We assumed that 90% of participants would have evidence of WMH at baseline and that approximately 64% would progress by one point or more on the RPS and that the mean progression score in the placebo group would be 1.293 at week 104 based on data from the Leukoaraiosis and Disability Study.²⁵ (link) We calculated, based on a Wilcoxon-Mann-Whitney test, that a sample size of 192 participants per group would give 80% power to detect a 30% relative reduction in progression score at a 5% significance level (nQuery Advisor® v7.0). This was chosen as a conservative minimally important difference as it is less than the previously reported difference seen with BP reduction.²⁶ (link) Further detail on the assumptions used of the sample size calculation are contained in the study protocol. We planned to randomize 232 participants per group to allow for withdrawals and for participants who would be unable to return for the week 104 MRI. We also calculated that 101 participants per group would be required to give 80% power at a 5% significance level to detect the previously reported 3.3 mmHg difference in change in SBP²⁶ (link) (assumed SD 8.3) at week 4.
All analyses were carried out according to the intention-to-treat (ITT) principle. Additional analyses were to be carried out using a per-protocol (PP) population. This excluded participants where there was an eligibility violation, participants who had more than 90 days of total treatment interruption and participants from one site where a serious breach of good clinical practice (GCP) was detected. The safety analysis set included all participants who received at least one dose of study medication.
The primary outcome was assessed by a linear regression model which adjusted for minimisation variables, site (as a random factor), and baseline characteristics associated with WMH progression (age, baseline National Institute of Health Stroke Scale score, baseline clinical SBP and Scheltens total score). Secondary outcomes were assessed by the same method except for progression on Schmidt's Progression score and presence of new brain infarction which were analysed by a Chi-squared test and logistic regression to adjust for minimisation variables. A p value of <0.05 was used for statistical significance. We pre-specified three sub-group analyses. These were by age, baseline uric acid level defined by the median and whether participation was completed before the introduction of Covid restrictions. We also performed a sensitivity analysis for MRI outcomes which included only those participants who had baseline and week 104 imaging performed on the same scanner, with the same sequence parameters, and no other quality issues deemed to affect interpretation.
The trial is registered in clinicaltrials.gov (registration number NCT02122718) and was adopted by the UK National Institute of Health Stroke Research Network and the Scottish Stroke Research Network.
The trial was overseen by a Trial Steering Committee (TSC) which met at least annually and comprised an independent chair, three other independent members, a participant representative, the Chief Investigator, and trial statistician. An independent Data Monitoring Committee (IDMC) met at least annually to review unblinded data. This comprised 4 independent members. The day-to-day running of the trial was overseen by the Trial Management Group at the University of Glasgow chaired by the Chief Investigator. Details of committee members are given in Appendix X.

Phage DNA was extracted from the purified high-titer phage suspension using the phenol–chloroform method described previously [6 (link),33 (link)]. The genomic DNA was sequenced using the Illumina HiSeq 4000 platform by Shanghai Majorbio Bio-pharm Technology Co., Ltd. After quality control and trimming, a total of 2,192,371,253 bp clean reads were obtained. Afterward, the clean reads were assembled using IDBA-UD version 1.1.1 to generate the final complete genome sequence [35 (link)].