CLIP实验方法


CLIP-seq早已是被广泛使用的protein-RNA结合互作位点研究的方法,但我们自己做的非常少,除了实验室老一辈人有的做过,现在的同学都没人做过,所以只好自己上,钻研一下CLIP的实验方法。

有些抑制剂和磷酸酶,如果没有,可尝试不加。

参考: Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq

Overview of CLIP method Overview of seCLIP protocol

Reagents

  • Dulbecco’s PBS (DPBS):细胞解离前,需采用不含钙和镁的制剂从培养物中冲洗掉螯合剂。
  • Lysis:
  • high-salt wash buffer
  • wash buffer
  • DNase
  • RNase I, 100 U/μl (Invitrogen, cat. no. AM2295)
  • murine RNase inhibitor
  • FastAP alkaline phosphatase(碱性磷酸酶). 1 U/μl (Thermo Scientific, cat. no. EF0652)
  • 10× PNK7 buffer. PNK buffer contains 700 mM Tris-HCl pH 7 and 100 mM MgCl2. This buffer is stable at −20 °C for ≥6 months.
  • PEG 8000

protocol step by step

Sample preparation and UV crosslinking (fig2a)

Timing:2-3h

  1. 贴壁细胞用DPBS洗一遍,然后添加足够预冷的DPBS覆盖单层细胞(统计细胞数量)
  2. 将crosslinker的金属块预冷后,放回crosslinker
  3. 将培养皿水平放到预冷金属块上,打开盖子,进行交联
    • irradiation: 254nm
    • quantity: 400mJ/cm2 once
  4. 交联完毕,马上用移液枪刮吹细胞,转移到离心管中,再用DPBS洗一遍,将剩余细胞转移到离心管中
  5. 4°C 300g离心3min,去上清,再用DPBS重悬细胞,至20 million cells/ml(或者其他理想浓度)
  6. 将细胞悬液按照理想的细胞数量,进行分装。分装后,4°C 300g离心3min,去上清。若不立即进行后续操作,可冻存于-80°C

Pause point:Cross-linked cells or tissue can be used immediately for lysis and IP or stored at −80 °C until use.


Bead preparation

以下步骤均需预冷的buffer
提前将lysis, high-salt wash and wash buffers预冷

Timing:1h

  1. 用500 ul预冷的lysis buffer wash beads 2次。注意不要震荡。弃去buffer,用100 ul lysis buffer重悬beads
  2. 每个IP样品,向washed beads中添加10ug的RBP-specific antibody,在rotator上室温混匀45min

10ug对于很多抗体是适宜的量,当然可以进行优化


Cell lysis, RNase digestion and IP

Timing:3h or overnight

  1. 向预冷的lysis buffer,加入蛋白酶抑制剂。用1ml的proteinase inhibitor+cold lysis buffer混匀细胞。将管子放在冰上裂解5min
  2. 在4°C下,超声仪低功率处理样品5min,30S on 30S off。样品置于冰上
  3. 在冰上,用DPBS以1:25的比例稀释RNase I。提前预热热混仪到37°C。向裂解样品中,加入5 ul Turbo DNase和10 ul的diluted RNase I,混匀。立即放到预热到37°C的热混仪上,以1,200 rpm的晃动频率,孵育5min整,然后置于冰上。立即加入11ul的murine RNase inhibitor。Centrifuge at 15,000g for 10 min at 4 °C.
  4. 离心的同时,用500ul的lysis buffer清洗两次上述准备好的beads。清洗后,离心除去剩余的wash buffer。将cleared lysate转移到antibody-bound beads,注意不要吸到细胞残片。4°C rotate 2h或者过夜(建议过夜)。

Dephosphorylation of IP samples

Timing: 1h

  1. 将lysate颠倒混匀。每个lysate(包括beads),转移20ul到两个新管子里,放在冰上暂存,待用(直到step23)。这部分用于做size-matched input samples (SMInput),一份用于跑RNA gel,一份用于做western blot
  2. 用磁力架,将beads和lysate分离开,并用500ul high-salt wash buffer洗两遍beads。为了防止盐浓度的突然变化破坏antibody-RBP复合物间的相互作用,采用过渡清洗的方法:加入500ul high-salt wash buffer,混匀,再加入500ul的wash buffer。先这样洗两次。再用wash buffer洗三次。
  3. 轻柔地spin down beads,除去残留的wash buffer。用dephosphorylation master mix重悬beads,轻弹管身,混匀。dephosphorylation master mix配方如下(每个样品的量50 ul):
Component Amount Final
H2O 38 -
10× FastAP Buffer 5 1x
Murine RNase inhibitor 2 80U
Turbo DNase 2 4U
FastAP enzyme 3 3U
Total 50 -
  1. 将反应体系,在37°C热混仪上,1,200 rpm摇10 min。这一步反应会去掉RNase I切割后,留在3’的磷酸基团。在孵育的同时,准备PNK master mix(下表是每个样品的量):
Component Amount (ul) Final
H2O 126 -
10× PNK7 buffer 20 1x
T4 PNK enzyme 4 40 U
total 150 ul
  1. 不需要去除 dephosphorylation mix,直接加入PNK master mix,继续在37°C热混仪上摇20 min。T4 PNK确保RNA片段3’ end完全去磷酸化,准备好3’ end ligation.
  2. 加200 ul high-salt wash buffer,混匀,磁性分离beads和上清,去除上清。继续使用过渡清洗的办法,先加500 ul high-salt wash buffer,混匀,再加入500 ul wash buffer,除去上清。然后用500 ul wash buffer洗三次。

❗CRITICAL STEP:
(Optional) Before carrying out 3′ ligation in the next steps, reserve 10% of the IP samples for biotin labeling to visualize the RNA cross-linked to your RBP of interest (see Box 1 for further explanation).


3’ RNA adapter ligation of IP samples

  1. 在室温下,准备3’ RNA adapter master(每个样品用量):
Component Amount (ul) Final
H2O 8.4 -
10× RNA ligase buffer (no DTT) 3.0 1.2x
0.1 M ATP 0.3 1.2 uM
100%DMSO 0.9 3.6%
1% (vol/vol) Tween-20 0.6 0.024%
50% (wt/vol) PEG 8000 9.0 18%
Murine RNase inhibitor 0.4 0.8 U
T4 RNA ligase high-concentration enzyme 2.4 72 U
Total 25 -

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