参考1：METTL16 promotes liver cancer stem cell self-renewal via controlling ribosome biogenesis and mRNA translation
参考2：abcam: RNA immunoprecipitation (RIP) protocol
material
lysis buffer
RNase inhibitor
protease inhibitor
RIP buffer (RNase-free reagent)
protocol (step by step)Cell lysate如果是做RIP-seq，为了增强RNA-protein互作，应做UV cross-linking
细胞裂解液配方 (native lysis buffer (RNA) )
sonication for shearing chromatin
IP前，取10%做input
Bead preparationRNA immunoprecipitationWashing off unbound materialDijest proteinsRNA purification用TRIzol RNA extraction reagent（1ml）重悬beads，然后提取RNA
加糖原。
Reverse transcription (RT) to cDNA and analysisXulab RIP protocolmaterial:
native lysis buffer + PMSF + proteinase inhibitor + RNase inhibitor
WB 150 (you can add RNase inhibitor)
protocol step by step:
prepare cells:1 x 10 cm dish (KI, WT), cold PBS washing
lysate cells:500 ul native lysis buffer (RNA) 4°C 30min.
centrifuge and isolate supernatant12,000g 10 min 4°C. Transfer supernatant into new tubes
keep inputkeep 2 x 50 ul supernatant for input (RNA and protein). 1ml TRIzol
prepare beadswash anti-flag beads (40ul) with 200ul washing buffer three times, and divide beads by 2:8 into two tubes
immunoprecipitate lysateadd left lysate into bead tubes as 2:8 ratio respectively, 4°C overnight on a rotator. 20% for RNA, 80% for protein.
discard immunodepletion and wash beads
centrifuge and keep supernatant as immunodepletion
wash beads with 200 ul washing buffer three times
RNA extractionAdd TRIzol into one tube for RNA extraction沉淀时先后加入异丙醇、1 ul糖原，冰上一个小时，或者-20过夜；reverse transcription using random primers
spoil proteinsAdd 1xSDS loading into another one to spoil beads for proteins