Blood mini kit

HBV DNA was extracted from 200 μl serum samples using Blood Mini Kit (Qiagen Inc., CA, USA). At baseline and each follow-up point, the quantification of HBV DNA was performed by Real-Time PCR using SYBR Green Master mix (Applied Biosystems [ABI], CA, USA) and primers F4 and R3 (Supplementary Table S6). The lower detection limit was 250 copies/ml.

was extracted from peripheral blood samples by a Qiagen DNA Blood Mini kit (Qiagen, Hilden, Germany)and quantitated by a Qubit Fluorometer (Life Technologies).

Samples were collected and treated in accordance with a pre-analytical guideline previously established by our group^{22 (link)}. In summary, blood was collected in EDTA K3 tubes and plasma was isolated within 2 hours. The isolation technique consist of a double centrifugation. Initially, tubes were centrifuged for 10 minutes at 4 °C and 1,200 g in a Heraeus Multifuge LR centrifuge. The supernatant was collected while carefully avoiding the buffy-coat. The second centrifugation was conducted for 10 minutes at 4 °C and 16,000 g. The supernatant was transferred to 1.5 ml tubes before performing the extraction of cirDNA or being stored at −20 °C. CirDNA extraction was performed with the Qiagen Blood Mini kit, following all steps of the protocol. In all, 0.2 to 1 ml of plasma was extracted in several successive passes on a column. The final elution volume was 80 to 130 µl and eluates were frozen at −20 °C prior to analysis by qPCR. Freeze-thaw cycles should be avoided to reduce the phenomenon of cirDNA fragmentation and the extracts are not kept longer than 3 months at −20 °C.

Intravenous SHIV challenge was conducted at least 200 days following autologous transplant, using challenge stocks identical to those for which historical data is referenced.^{36 (link),38 (link)} Blood draws were collected by venipuncture into heparin or EDTA collection tubes. Viral load and CD4⁺ T-cell counts were measured at baseline (pre-SHIV), and 1–4 times per month following challenge, as described previously.^{56 (link),57 (link)} “Normal” CD4⁺ T-cell counts were calculated by averaging 2–5 weekly measurements from 26 healthy, SHIV-naive animals, and displayed as a range ±1 SD (Figure 4b). Total leukocytes were collected by hemolysis of whole blood and peripheral blood mononuclear cells (PBMC) were collected by ficoll gradient centrifugation. Total genomic DNA was prepared from each source by Blood Mini Kit (Qiagen, Hilden, Germany) or Masterpure DNA Purification Kit (Epicentre Biotechnologies, Madison, WI). Cal-1 gene marking from total genomic DNA samples was measured by Taqman as described previously.^{27 (link)}

DNA was extracted by Qiagen Blood mini kit (Carlsbad, CA) from the collected blood specimens. Microscopy results were confirmed by both real time PCR and nested PCR [18 (link), 19 (link)]. If any discrepancies were found by different diagnostic methods, those samples were excluded from the study.