Total RNA (600 ng) was ligated to 5 μM of 5′-adenylated, 3′-blocked adaptor (Universal miRNA Cloning Linker, New England BioLabs) with 280 units of T4 RNA ligase, Truncated KQ (New England BioLabs), 25% PEG 8000 and 1 μl of RNaseOUT (Life Technologies) in a 20-μl reaction at 25 °C for 16–24 h. After cleanup with RNA Clean and Concentrator columns (Zymo Research), followed by DNase treatment, cDNA was synthesized with 5 pmol of universal RT primer (Supplementary Table 6 ) and SuperScript III reverse transcriptase. PCR amplification was carried out using 5 μM of the TERC_L2 and universal RT or TERC_L3 and universal RT primer sets (Supplementary Table 6 ) with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). PCR products were directly analyzed on 2.5% agarose gels to visualize mature TERC and extended TERC transcripts or subjected to QIAquick PCR purification columns (Qiagen) for library preparation for deep sequencing. For Sanger sequencing, 3′ RACE PCR products were directly cloned into the pCR4_TOPO vector (Life Technologies), and individual clones were sequenced using the TERC_L2 or TERC_L3 primer.
Truncated kq
Manufactured by New England Biolabs
Truncated KQ is a protein fragment that retains the key functional domains of the full-length protein. It is often used in research applications that require a simplified version of the original protein.
Automatically generated - may contain errors
Lab products found in correlation
T4 rna ligase, by Thermo Fisher Scientific (2 mentions)
T4 rna ligase 2, by New England Biolabs (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
T4 rna ligase, by New England Biolabs (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Universal mirna cloning linker, by New England Biolabs (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Rna clean and concentrator column, by Zymo Research (1 mentions)
Qiaquick pcr purification column, by Qiagen (1 mentions)
4 protocols using truncated kq
1
RNA-seq Analysis of TERC Transcripts
2
BrdU-CLIP cDNA Library Preparation
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The eluted RNA from the m6A-IP step and the input RNA fragments were first treated with T4 PNK to remove its 3′ phospho group and then ligated to 68 pmol preadenylated DNA linker (L32N from IDT) with T4 RNA ligase 2 and truncated KQ (New England Biolabs, catalog no. M0373L) overnight at 16°C. This ligation mixture was subject to 8% PAGE purification to harvest the ligated product. The purified RNAs from m6A-IP and m6A-CLIP and their input RNA fragments were all subjected to the same BrdU-CLIP cDNA library preparation. The detailed BrdU-CLIP cDNA library protocol was described previously (Weyn-Vanhentenryck et al. 2014 (link)), and we practiced a similar procedure with an improved RT primer set that starts with three “D” (no C) nucleotides on the 5′ end and improves ligation efficiency.
3
Small RNA Sequencing Protocol
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Small RNA libraries were generated as described previously with slight modifications (McGinn and Czech 2014 (link)). Briefly, 18- to 29-nt sized small RNAs were purified by PAGE from 12 µg of total RNA from ovaries. Next, the 3′ adapter (containing four random nucleotides at the 5′ end) (Jayaprakash et al. 2011 (link)) was ligated using T4 RNA ligase 2 and truncated KQ (New England Biolabs). Following recovery of the products by PAGE purification, the 5′ adapter (containing four random nucleotides at the 3′ end) was ligated to the small RNAs using T4 RNA ligase (Ambion). Small RNAs containing both adapters were recovered by PAGE purification, reverse-transcribed, and PCR-amplified. Libraries were sequenced on an Illumina HiSeq 4000 (Illumina).
4
Small RNA Library Generation Protocol
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Small RNA libraries were generated as described previously with slight modifications (McGinn and Czech 2014 (link)). Briefly, 18- to 29-nt-long small RNAs were purified by PAGE from 15 µg of total RNA from ovaries or OSCs. Next, the 3′ adapter (containing four random nucleotides at the 5′ end) (Jayaprakash et al. 2011 (link)) was ligated using T4 RNA ligase 2 and truncated KQ (New England Biolabs). Following recovery of the products by PAGE purification, the 5′ adapter (containing four random nucleotides at the 3′ end) was ligated to the small RNAs using T4 RNA ligase (Ambion). Small RNAs containing both adapters were recovered by PAGE purification, reverse-transcribed, and PCR-amplified. Libraries were sequenced on an Illumina HiSeq 4000. All adapter sequences are in Supplemental Table S6 .
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