銆愮敘鍝佺法铏熴��2793951
銆愯嫳鏂囧悕绋便��5fC CLEVER-seq Kit
銆愪腑鏂囧悕绋便��鍏ㄥ熀鍥犵祫5-閱涘熀鑳炲槯鍟跺柈绱拌優妾㈡脯瑭﹀姂鐩�
銆愮敘鍝佸収瀹广��
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鐢㈠搧绲勬垚
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鍚噺
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婧舵恫P1锛�Buffer P1锛�
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2.4 mL
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M
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24绠�
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妯″紡搴忓垪
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24ul
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娉細婧舵恫P1锛氫笝浜岃厛鍙嶆噳娑�锛�M锛氫笝浜岃厛
銆愪繚瀛樻浠躲��妯″紡搴忓垪缃簬-20 °C锛屽彲淇濆瓨12鍊嬫湀銆�
M鍜屾憾娑�P1缃簬4 °C锛屽彲淇濆瓨12鍊嬫湀銆�
M闇�閬垮厜淇濆瓨銆�
銆愮敘鍝佹杩般��
5-鐢插熀鑳炲槯鍟朵慨椋撅紙5mC锛夋槸鐪熸牳绱拌優鍩哄洜绲勪腑鏈�鐐洪噸瑕佺殑琛ㄨ閬哄偝淇＞涔嬩竴銆�5mC鍙互鍦�TET瀹舵棌閰剁殑浣滅敤涓嬮�愭姘у寲鐢熸垚5-缇ョ敳鍩鸿優鍢у暥锛�5hmC锛�銆�5-閱涘熀鑳炲槯鍟讹紙5fC锛夊拰5-缇у熀鑳炲槯鍟讹紙5caC锛�锛涘皪浜庝笂杩板咕绋柊鍨�DNA淇＞鐨勫姛鑳界爺绌舵槸鐩墠鏍搁吀琛ㄨ閬哄偝瀛哥殑涓�鍊嬬爺绌剁啽榛�銆�
鐧鹃潏濞佹帹鍑烘柊鍨嬪柈绱拌優5fC妾㈡脯瑭﹀姂鐩�锛屽畠涓昏鐢ㄤ簬鍠窗鑳�5-閱涘熀鑳炲槯鍟� (5-formylcytosine, 5fC锛夌殑鍏ㄥ熀鍥犵祫鍠牽鍩哄垎杈ㄧ巼娓簭锛涗篃鍙噳鐢ㄤ簬5fC鍦ㄦ棭鏈熻儦鑳庣櫦(f膩)鑲插拰鐤剧梾鐧�(f膩)鐢熺櫦(f膩)灞曢亷绋嬬殑鐢熺墿瀛稿姛鑳界爺绌�銆�
銆愯嚜鍌欐潗鏂欍��
• Oligo Clean & Concentrator (ZYMO, #D4060)
• NEBNext® Ultra™ II DNA Library Prep Kit for Illumina锛�NEB锛岋純E7645锛�
• NEBNext Singleplex or Multiplex Oligos for Illumina (NEB #E7350, #E7335, #E7500, #E7600, #E6609, #E7710, or #E7730)
• MALBAC Single Cell WGA Kit锛�Yikon Genomice Co., Ltd Cat.No.YK001A/B锛�
• AMPure XP Beads锛�Beckman Coulter, Inc. #A63881锛�
• 鐒℃按涔欓唶
• 婊呰弻瓒呯磾姘�
• 浣庡惛闄�EP銆�PCR绠�
• 纾佸姏鏋�
• PCR鐔卞惊鐠�(hu谩n)鍎�
• Thermo-mixer (Eppendorf)
銆愭祦绋嬫杩般��
5fC CLEVER-seq Kit 鍘熺悊鍦�
銆愭敞鎰忎簨闋呫��
A. 纾佺彔鎿嶄綔娉ㄦ剰浜嬮爡锛�
• 鎳夊皣纾佺彔瀛佃偛鑷冲婧悗鍐嶄娇鐢�
• 姣忔鍚稿彇纾佺彔鍓嶉兘鎳夊皣鍏跺厖鍒嗘贩鍕�
• DNA妯ｅ搧鍔犲叆纾佺彔鍚庢噳灏囧叾鍏呭垎娣峰嫽
• 鎵�鏈夌鐝犳搷浣滈兘鎳変簬瀹ゆ韩閫茶
• 绉诲彇涓婃竻鎿嶄綔鎳夊湪纾佺彔琚竟搴曞惛闄勫悗灏忓績閫茶
• 纾佺彔鍦ㄦ礂鑴箣鍓嶆噳鍏呭垎骞茬嚗,閬垮厤涔欓唶娈樼暀褰遍熆鍚庣簩(x霉)瀵﹂
B. 閬垮厤妯ｅ搧浜ゅ弶姹℃煋
• 鍚稿彇涓嶅悓妯ｅ搧鏅傛洿鎻涙闋�
C. 閰剁殑鍙栨斁鎳夊湪鍐颁笂閫茶锛岀偤閬垮厤鍙嶅京鍑嶈瀺鎴栭暦鏈熶娇鐢ㄥ悗娲绘�т笅闄�,鎴戝�戞帹钖︽偍鍦ㄩ娆′娇鐢ㄥ悗灏囧墿浣欒│鍔戝皬浠藉垎瑁濆噸瀛�銆�
D. 鍠窗鑳為伩鍏嶆薄鏌�
•鍠窗鑳炴搷浣滃井閲�DNA锛屽湪闋愭摯澧炲墠锛屾搷浣滈渶鍦ㄨ秴鍑堝伐浣滆嚭涓�茶锛岄伩鍏嶆薄鏌�
•婧舵恫鍚稿彇闇�浣跨敤甯舵烤鑺殑妲嶉牠
E. 闋愭摯澧炲墠鎵�鏈夊弽鎳夐渶鍦ㄤ竴鍊�EP绠′腑閫茶
銆愬椹楁椹熴��
璧峰鏉愭枡锛氬皣鍒嗛伕濂界殑涓�鍊嬪柈绱拌優缃簬MALBAC Single Cell WGA Kit 锛�Yikon Genomice Co., Ltd Cat.No.YK001A/B锛夌殑Cell Lysis Buffer涓��
涓�. 绱拌優瑁傝В
鎸夌収MALBAC Single Cell WGA Kit锛�Yikon Genomice Co., Ltd Cat.No.YK001A/B锛夎鏄庢浉閫茶绱拌優瑁傝В銆�
浜�. M浠嬪皫鐨�5fC妯欒
鍦ㄨ瑙ｇ殑绱拌優涓姞鍏�1ul M锛� 1ul婧舵恫P1鍜�1ul妯″紡搴忓垪锛屽厖鍒嗘贩鍕�锛岀疆浜�Thermo-mixer (Eppendorf)涓紝37°C锛�850rmp閬垮厜鍙嶆噳20h銆�
涓�. 鍠窗鑳炴摯澧�
1. 灏囨瑷樺畬鎴愮殑绱拌優鎸夌収MALBAC Single Cell WGA Kit锛�Yikon Genomice Co., Ltd Cat.No.YK001A/B锛夎鏄庢浉閫茶鍠窗鑳炴摯澧�銆�
2. 鎿村瀹屾垚鍚�锛屾寜鐓�Oligo Clean & Concentrator (ZYMO, #D4060)瑾槑鏇哥磾鍖栦笂杩板畬鎴愭摯澧炵殑鍠窗鑳炪��
娉細鍔犲叆30ul 娲楄劔绶╂矕娑叉礂鑴�銆�
鍥涳紟鏂囧韩妲嬪缓
鎸夌収NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB锛岋純E7645)瑾槑鏇搁�茶寤哄韩鎿村銆�