Patent SG-11201811048U-A (SG)
Title: Single-cell transcript sequencing
Inventor: LIVAK KENNETH J
IPC: C12Q1/68
Filed: 2017-07-12
Abstract: INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property MD 1101111 0 DOI HIM 011101 0 0111111 01110011111111111111111111111111111111 Organization International Bureau (10) International Publication Number (43) International Publication Date ......0\" WO 2018/013723 Al 18 January 2018 (18.01.2018) W I P0 I P C T (51) International Patent Classification: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, C12Q 1/68 (2006.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (21) International Application Number: KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, PCT/US2017/041770 MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 12 July 2017 (12.07.2017) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (30) Priority Data: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 62/362,425 14 July 2016 (14.07.2016) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, 62/463,487 24 February 2017 (24.02.2017) US EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, (71) Applicant: FLUIDIGM CORPORATION [US/US]; TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, 7000 Shoreline Court, Suite 100, South San Francisco, Cal- KM, ML, MR, NE, SN, TD, TG). ifornia 94080 (US). (72) Inventors: LIVAK, Kenneth J.; 7000 Shoreline Court, Declarations under Rule 4.17: Suite 100, South San Francisco, California 94080 (US). — of inventorship (Rule 4.17(iv)) — FEKETE, Richard A.; 7000 Shoreline Court, Suite 100, Published: South San Francisco, California 94080 (US). — with international search report (Art. 21(3)) = (74) Agent: HALIDAY, Emily M. et al.; Weaver Austin Vil- leneuve & Sampson LLP, P.O. Box 70250, Oakland, Cali- = fornia 94612-0250 (US). = Designated States (unless otherwise indicated, for every = (81) kind of national protection available): AE, AG, AL, AM, — AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, = = (54) Title: SINGLE-CELL TRANSCRIPT SEQUENCING = = 1 = = = I I = Fresh = = Fixed — ,-1 .4 ei N IN Figure 10 M ,-1 © (57) : Described herein are methods for preparing DNA templates for single-cell transcript sequencing of RNA from a popu- P i) \" lation of cells. The methods entail distributing cells from the population into separate reaction volumes so that a plurality of separate Il reaction volumes each contain a single, isolated cell, wherein the cells have been treated with a fixative prior to distribution. The isolated cells are then permeabilized or disrupted, and cDNA is prepared by reverse transcript, followed by amplification. Also provided is a — novel chemistry for efficient production of DNA templates from T-cell receptors or immunoglobulins in single cells.