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 preparation
RNA immunoprecipitation
Washing off unbound material
Dijest proteins
RNA purification
用TRIzol RNA extraction reagent（1ml）重悬beads，然后提取RNA
加糖原。
Reverse transcription (RT) to cDNA and analysis
Xulab RIP protocol
material:
- 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 supernatant
12,000g 10 min 4°C. Transfer supernatant into new tubes - keep input
keep 2 x 50 ul supernatant for input (RNA and protein). 1ml TRIzol - prepare beads
wash anti-flag beads (40ul) with 200ul washing buffer three times, and divide beads by 2:8 into two tubes - immunoprecipitate lysate
add 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 extraction
Add 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 proteins
Add 1xSDS loading into another one to spoil beads for proteins
