Section summary #

Here I attempted to prepare 1 replicate of T7Init Mix 1 with different enzymatic treatments. Fred pointed out that I should use chloroquine gels to confirm topological manipulations where successful. Repeated chloroquine gels showed Topoisomerase treatments to be effective at relaxing the plasmid mix but Gyrase left something to be desired compared to Stoltz et al. Since Gyrase was causing headaches I decided to drop it from this first go around of sequencing.

During my first attempt with these samples bisulfite treatment and PCR amplification succeeded but I lost basically all of my samples at the agarose gel extraction step. I re-did the PCR reaction with my remaining bisulfite converted samples but this time the PCR failed for unknown reasons.

Ultimately this replicate yielded no usable samples.

Substrate preparations #

3/27/28 #

Four samples were prepared from T7InitMix-0 which contains approximately 10 ng / ul / construct. Volumes of each construct added were calculated based on appearance of 1 ul of construct on agarose gel compared to pFC53 and pFC9 (see experiment details here).

The four samples and described in the section below.

0: Supercoiled #

Reagent	Volume (ul)
npH20	92
T7InitMix-1	8

Incubated 37C 1 hour.

1: BamHI linearized #

Reagent	Volume (ul)
npH20	80
T7InitMix-1	8
NEB BamHI-HF	2
NEB rCutSmart 10X Buffer	10

Incubated 37C 1 hour.

2: Hyper-supercoiled (Gyrase treated) #

Reagent	Volume (ul)
npH20	38
T7InitMix-1	8
Topogen 5x Gyrase Assay Buffer	12
TopoGen Gyrase Enzyme	2

Incubated 37C 1 hour.

3: Relaxed (Topoisomerase I treated) #

Reagent	Volume (ul)
npH20	80
T7InitMix-1	8
NEB 10x rCutSmart Buffer	10
NEB TopoisomeraseI	2

Incubated 37C 20 mins.

After completing incubations all samples were phenol/chloroform extracted using homemade phase lock columns and then EtOH precipitated and re-suspended in 20 ul 10mM Tris-HCl. Samples were then stored at -20C.

Reagent details #

Reagent cat and lot numbers for the reactions described above are listed in the table below.

Reagent details ↕

Reagent	Cat number	Lot number
NEB rCutSmart 10x Buffer	B60042	10132778
Topogen Ecoli DNA Gyrase 5x Assay Buffer	T62000G-1	22JA25
Topogen Ecoli DNA Gyrase 100 units	T62000G-1	22JA25
NEB TopoisomeraseI (E.coli)	M0301S	10123119
NEB BamHI-HF	R31362	10508

Nanodrop results #

After completing all treatments and precipitations nanodroped samples to confirm still have DNA.

Sample	ng/ul	260/280	260/230
0. Supercoiled	267.5	1.8	2.035
1. Linearized	357.2	1.8	2.06
2. Hyper-supercoiled	290.5	1.732	1.772
3. Relaxed	268.3	1.745	2.067

IVT with T7 and bisulfite conversion #

3/28/22 #

Prior to IVT all samples were spiked with 40 ng of pFC9 as a positive control. Samples were then treated according to IVT + Bisulfite treatment protocol.

Sample 0 was treated according to “IVT followed by bisulfite conversion for SMRF-seq samples requiring untranscribed controls” while others were treated according to “IVT followed by bisulfite conversion for SMRF-seq samples not requiring untranscribed controls”.

Sample table #

Following samples described in the table below were produced. Included along with nanodrop results after completing bisulfite conversion.

Also available in this document.

Sample table and nanodrop results

Sample Series	Sample Number	Formal name	Transcribed	TopoisomeraseI	Gyrase	BamHI-HF	RnaseA	RnaseH	Deprotienzied	ng/ul	260/280	260/230	Name
EHS0	0	EHS0-R0	FALSE	FALSE	FALSE	FALSE	FALSE	FALSE	FALSE	3.6	1.16	0.457	T7 SC Txn -
EHS0	1	EHS0-R1	TRUE	FALSE	FALSE	FALSE	FALSE	FALSE	FALSE	39	2.019	2.202	T7 SC Txn +
EHS0	2	EHS0-R2	TRUE	FALSE	TRUE	FALSE	FALSE	FALSE	FALSE	27.8	2.17	1.057	T7 Gyr. Txn+ 2
EHS0	3	EHS0-R3	TRUE	FALSE	TRUE	FALSE	FALSE	FALSE	FALSE	15.6	3.395	3.756	T7 Gyr. Txn+ 1
EHS0	4	EHS0-R4	TRUE	TRUE	FALSE	FALSE	FALSE	FALSE	FALSE	29.2	2.214	2.133	T7 Topo Txn+ 1
EHS0	5	EHS0-R5	TRUE	TRUE	FALSE	FALSE	FALSE	FALSE	FALSE	29.9	2.62	2.006	T7 Topo Txn+ 2
EHS0	6	EHS0-R6	TRUE	FALSE	FALSE	TRUE	FALSE	FALSE	FALSE	49.3	2.117	2.372	T7 Lin. Txn+ 1
EHS0	7	EHS0-R7	TRUE	FALSE	FALSE	TRUE	FALSE	FALSE	FALSE	48.7	2.196	1.446	T7 Lin. Txn+ 2
EHS0	8	EHS0-R8	TRUE	FALSE	FALSE	FALSE	TRUE	FALSE	TRUE	5.4	1.387	0.453	T7 DP SC Txn+
EHS0	9	EHS0-R9	TRUE	FALSE	TRUE	FALSE	TRUE	FALSE	TRUE	1.3	0.713	0.138	T7 DP Gyr. Txn+ 1
EHS0	10	EHS0-R10	TRUE	FALSE	TRUE	FALSE	TRUE	FALSE	TRUE	9.4	3.22	1.467	T7 DP Gyr. Txn+ 2
EHS1	11	EHS1-R11	TRUE	TRUE	FALSE	FALSE	TRUE	FALSE	TRUE	-0.5	-0.486	0.988	T7 DP Topo. Txn+ 1
EHS2	12	EHS2-R12	TRUE	TRUE	FALSE	FALSE	TRUE	FALSE	TRUE	-0.2	-0.275	-0.032	T7 DP Topo. Txn+ 2
EHS3	13	EHS3-R13	TRUE	FALSE	FALSE	TRUE	TRUE	FALSE	TRUE	0.6	0.543	0.084	T7 DP Lin. Txn+ 1
EHS4	14	EHS4-R14	TRUE	FALSE	FALSE	TRUE	TRUE	FALSE	TRUE	2.6	1.336	0.344	T7 DP Lin. Txn+ 2
EHS5	15	EHS5-R15	TRUE	FALSE	FALSE	FALSE	TRUE	TRUE	TRUE	-4.6	-0.482	-0.124	T7 DP SC RH1 Txn+

While sample values were quite low (even for bisulfite) I think it is still worth moving forward with the PCR reactions to really see if anything is in those tubes. What was surprising was that proteinized samples tended to have much higher specs. This may have been due to the lack of RnaseA digestion with these samples though.

Reagent details #

Transcription and Bisulfite conversion reagents

Reagent	Cat Number	Lot Number	Notes
Zymogen lightning conversion reagent	D5030-1	211521	Used for treating protienized transcribed samples and supercoiled un-transcribed control
Zymogen lightning conversion reagent	D5030-1	211521	Used for treating de-protienized samples
Zymogen EZ DNA Methylation lightning kit	11-338	ZRC202947
NEB RnaseH buffer 10x	B0297S	10085445
NEB RNA Pol Reaction Buffer 10x	B9012S	10073286
Thermo Scientific RNA grade glycogen	R0551	1191453
NEB rNTP Mix	N0466S	10085868

3/29/22 #

PCR of bisulfite converted samples #

Formal name	Name	Transcribed	TopoisomeraseI	Gyrase	BamHI-HF	RnaseA	RnaseH	Deprotienzied	ng/ul	260/280	260/230
EHS0-R0	T7 SC Txn -	FALSE	FALSE	FALSE	FALSE	FALSE	FALSE	FALSE	3.6	1.16	0.457
EHS0-R1	T7 SC Txn +	TRUE	FALSE	FALSE	FALSE	FALSE	FALSE	FALSE	39	2.019	2.202
EHS0-R6	T7 Lin. Txn+ 1	TRUE	FALSE	FALSE	TRUE	FALSE	FALSE	FALSE	49.3	2.117	2.372
EHS0-R7	T7 Lin. Txn+ 2	TRUE	FALSE	FALSE	TRUE	FALSE	FALSE	FALSE	48.7	2.196	1.446
EHS0-R8	T7 DP SC Txn+	TRUE	FALSE	FALSE	FALSE	TRUE	FALSE	TRUE	5.4	1.387	0.453
EHS3-R13	T7 DP Lin. Txn+ 1	TRUE	FALSE	FALSE	TRUE	TRUE	FALSE	TRUE	0.6	0.543	0.084
EHS4-R14	T7 DP Lin. Txn+ 2	TRUE	FALSE	FALSE	TRUE	TRUE	FALSE	TRUE	2.6	1.336	0.344
EHS5-R15	T7 DP SC RH1 Txn+	TRUE	FALSE	FALSE	FALSE	TRUE	TRUE	TRUE	-4.6	-0.482	-0.124

Sample barcoded primer assignment #

Sample Name	Formal name	Forward Primer Name	Oligo ID	Barcode
T7 SC Txn -	EHS0-R0	PacBioBarFwd-0	oEH30	CGGTTAGA
T7 SC Txn +	EHS0-R1	PacBioBarFwd-1	oEH31	CCCTCTTT
T7 Lin. Txn+ 1	EHS0-R6	PacBioBarFwd-2	oEH32	CGTTTCTG
T7 DP SC Txn+	EHS0-R8	PacBioBarFwd-3	oEH33	ATGGATCG
T7 DP Lin. Txn+ 2	EHS4-R14	PacBioBarFwd-4	oEH34	GGTAACAC
T7 DP SC RH1 Txn+	EHS5-R15	PacBioBarFwd-5	oEH35	CAAGCAGA

Table link.

PCR reaction details #

Total Volume	5x Phusion GC buffer	10 mM dNPTS	10 uM Primer Mix	Bisulfite converted template	PhuU Polymerase	npH20
30	6	0.4	1.5	4	0.3	18.1

Reactions were assembled from master mix and then primers and template were added to each reaction aqliuote.

All samples were run for 15 PCR cycles. Annealed at 58.9C for 30 seconds with 2:30 extension time at 72C. Samples then held at 4C for ~2 hours.

Agarose gel extraction PCR products #

Imaged using phone camera on blue light transilluminator.

Preformed gel extraction according to Zymogen protocol. Elluted extracted products in 30 ul ellution buffer (10 mM Tris HCl).

Nanodrop extracted products #

Agarose gel extracted products #

Sample	ng_ul	260_280	260_230
1	12.4	2.299	0.019
2	9	2.26	0.022
3	5.5	2.767	0.011
4	8.9	2.291	0.015
5	7.8	2.375	0.011
6	18.8	1.822	0.047

Testing chloroquine gel protocol using T7Init Mix 0 +- Topo treatment #

Agarose gel image #

3/30/22 #

Repeat PCR for agarose gel extraction #

PCR reaction was done with same protocol preformed previously with these samples. Ran PCR overnight to run on gel in the morning.

3/31/22 #

Agarose gel extraction of 3/30 PCR products #

Ran complete 30 ul reaction of each sample on agarose gel (1x TAE 60V 2 hrs) and viewed on the trans illuminator (no gel image). However, even though the postive control (using T7InitMix 1 unconverted template) showed a band all bisulfite treated samples except the un-transcribed control did not show a band. Not sure what happened here but seems to be something specific to transcribed reactions and not the PCR master mix. Unfortunately this replicate ends here because of this failed PCR.